Compositions of and methods of using sulfatases from Flavobacterium heparinum

ABSTRACT

This invention is related, in part, to sulfatase enzymes and methods of their use.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/006,794, filed Jan. 4, 2008, which claims priority under 35 U.S.C. §119 from U.S. provisional application Ser. No. 60/879,272, filed Jan. 5, 2007. The entire contents of both prior applications are herein incorporated by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant number GM 57073 awarded by the National Institutes of Health. Accordingly, the government has certain rights in the invention.

FIELD OF THE INVENTION

This invention is related, in part, to sulfatase enzymes and methods of their use.

BACKGROUND OF THE INVENTION

Heparan sulfate glycosaminoglycans (HSGAGs) comprise an important polysaccharide constituent of many proteoglycans (Bernfield et al., 1999 Annu Rev Biochem 68, 729-777). These glycans are linear polymers based on the variably repeating disaccharide unit (uronic acid α/β1→4 glucosamine)_(n), where n represents a variably repeating number (typically 10-200). As present in nature, these sugars possess an extensive chemical heterogeneity which is largely attributed to the mosaic arrangement of O- and N-linked sulfates present at different positions along each sugar chain (Esko et al., 2001 J Clin Invest 108, 169-173; Sasisekharan et al., 2002 Nat Rev Cancer 2, 521-528). Additional structural variations include the presence of N-linked acetates at the glucosamine C2 position as well as the epimerization of the uronic acid C5 carboxylate that distinguishes β-D-glucuronic acid from α-L-iduronic acid. Fundamental to understanding HSGAG structure-activity relationships is the appreciation that the polydispersity of glycan fine structure is not random. Instead, it is the end product of activities regulated in a cell and tissue specific fashion. This programmed diversity of HSGAG structure (Esko et al., 2001 J Clin Invest 108, 169-173) ultimately plays out at a functional level, namely through the dynamic regulation of numerous biochemical signaling pathways (Esko et al., 2001 J Clin Invest 108, 169-173) relating to such processes as cell growth and differentiation (Sasisekharan et al., 2002 Nat Rev Cancer 2, 521-528), cell death (Prince et al., 2002 Dev Dyn 223, 497-516; Lai et al., 2004 Gastroenterology 126, 231-248), intercellular communication, adhesion and tissue morphogenesis (Hacker et al., 2005 Nat Rev Mol Cell Biol 6, 530-541). HSGAGs (present as structurally-defined binding epitopes on the cell surface) also play an important role in microbial pathogenesis (Liu et al., 2002 J Biol Chem 277, 33456-33467; Vives et al., 2006 Curr Gene Ther 6, 35-44).

In contrast to the complex enzymatic process by which these polysaccharides are made, it appears that their catabolism is more straightforward, both in the scope of its purpose and the means by which it is carried out at the biochemical level. In the mammalian lysosome for example, GAG degradation follows an obligatory sequence of depolymerization steps, using enzymes which follow a predominantly exolytic mode of action. As such, the substrate specificity of one enzyme is largely predicated on the activity of the enzymes which precede it. Essential to this sequence are several sulfohydrolases which desulfate the sugar backbone as a prerequisite to the ensuing glycosidase step. These sulfatases are structure-specific enzymes, each one hydrolyzing a unique sulfate position within the heparin disaccharide repeat unit present at the non-reducing end.

Sequential GAG degradation is not unique to the eukaryotic lysosome. This process has been demonstrated in several microorganisms as well (Dietrich et al., 1973 J Biol Chem 248, 6408-6415; Nakamura et al., 1988 J Clin Microbiol 26, 1070-1071; Lohse et al., 1992 J Biol Chem 267, 24347-24355), which depend on sulfated polysaccharides not only as a carbon source but often as a means of scavenging inorganic sulfate (Kertesz, 2000 FEMS Microbiol Rev 24, 135-175). The gram-negative soil bacterium Flavobacterium heparinum (a.k.a. Pedobacter heparinus) is an excellent example of this process, having also proven to be a particularly rich biological source for the isolation and molecular cloning of several GAG-degrading enzymes (Sasishekaran et al., 1993 Proc Natl Acad Sci USA 90, 3660-3664; Godavarti et al., 1996 Biochem Biophys Res Commun 225, 751-758). Like the lysosomal pathway, many of the flavobacterial enzymes possess a high degree of substrate specificity.

SUMMARY OF THE INVENTION

The invention relates, in part, to sulfatase enzymes and polypeptides, nucleic acids that encode them, as well as compositions of the aforementioned molecules and methods of their use.

In one aspect a sulfatase enzyme is provided, wherein the sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 2, 4, 17 or 18. In one embodiment, the sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 4.

In another aspect of the invention, modified sulfatases are provided. In one aspect, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 2 or 17, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is the residue at position 39, 40, 80, 83, 127, 129, 206, 277, 386 or 503 (said positions are relative to the numbering of the residues in SEQ ID NO: 17). In another aspect, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 4 or 18, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is the residue at position 39, 80, 83, 128, 175 or 245 (said positions are relative to the numbering of the residues in SEQ ID NO: 18). In other embodiments, the at least one amino acid residue that has been substituted or deleted is any of the residues specifically listed herein, such as in the Examples or as identified by the alignment provided in FIG. 11. In still another aspect, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 2 or 17, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is not the residue at position 39, 40, 80, 83, 127, 129, 206, 277, 386 or 503. In a further aspect, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 4 or 18, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is not the residue at position 39, 80, 83, 128, 175 or 245. In further embodiments, the at least one amino acid residue that has been substituted or deleted is not any of the residues specifically listed herein.

In one aspect, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 2 or 17, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is the residue at position 40, 41, 80, 84, 128, 130, 206, 277, 387 or 504 (said positions are relative to the numbering of the residues in SEQ ID NO: 17). In one embodiment, residue 207 is substituted or deleted. In another embodiment, residue 276 is substituted or deleted. In another aspect, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 4 or 18, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is the residue at position 40, 80, 84, 129, 176 or 246 (said positions are relative to the numbering of the residues in SEQ ID NO: 18). In other embodiments, the at least one amino acid residue that has been substituted or deleted is any of the residues specifically listed herein, such as in the Examples or as identified by the alignment provided in FIG. 11. In still another aspect, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 2 or 17, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is not the residue at position 40, 41, 80, 84, 128, 130, 206, 277, 387 or 504 (said positions are relative to the numbering of the residues in SEQ ID NO: 17). In one embodiment, residue 207 has not been substituted or deleted. In another embodiment, residue 276 has not been substituted or deleted. In a further aspect, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 4 or 18, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is not the residue at position 40, 80, 84, 129, 176 or 246. In further embodiments, the at least one amino acid residue that has been substituted or deleted is not any of the residues specifically listed herein.

In another embodiment, the sulfatase enzyme comprises an amino acid sequence wherein the active site cysteine is in its modified form (FGLy).

In still another embodiment, the sulfatase enzymes provided are substantially pure. In a further embodiment, the sulfatase enzymes provided are isolated. In still a further embodiment, the sulfatase enzymes provided are recombinant.

In another aspect, a method is provided comprising producing or obtaining a modified sulfatase and determining an activity of the modified sulfatase. In one embodiment, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 2 or 17, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is the residue at position 39, 40, 80, 83, 127, 129, 206, 277, 386 or 503. In another embodiment, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 4 or 18, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is the residue at position 39, 80, 83, 128, 175 or 245. In still another embodiment, the at least one amino acid residue that has been substituted or deleted is any of the residues specifically listed herein. In a further embodiment, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 2 or 17, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is not the residue at position 39, 40, 80, 83, 127, 129, 206, 277, 386 or 503. In another embodiment, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 4 or 18, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is not the residue at position 39, 80, 83, 128, 175 or 245. In further embodiments, the at least one amino acid residue that has been substituted or deleted is not any of the residues specifically listed herein. In another embodiment, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 2 or 17, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is the residue at position 40, 41, 80, 84, 128, 130, 206, 277, 387 or 504 (said positions are relative to the numbering of the residues in SEQ ID NO: 17). In one embodiment, residue 207 is substituted or deleted. In another embodiment, residue 276 is substituted or deleted. In still another embodiment, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 4 or 18, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is the residue at position 40, 80, 84, 129, 176 or 246 (said positions are relative to the numbering of the residues in SEQ ID NO: 18). In another embodiment, the at least one amino acid residue that has been substituted or deleted is any of the residues specifically listed herein. In still another embodiment, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 2 or 17, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is not the residue at position 40, 41, 80, 84, 128, 130, 206, 277, 387 or 504 (said positions are relative to the numbering of the residues in SEQ ID NO: 17). In one embodiment, residue 207 has not been substituted or deleted. In another embodiment, residue 276 has not been substituted or deleted. In a further embodiment, the modified sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 4 or 18, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is not the residue at position 40, 80, 84, 129, 176 or 246. In further embodiments, the at least one amino acid residue that has been substituted or deleted is not any of the residues specifically listed herein.

In yet another embodiment, producing the modified sulfatase includes the step of modifying a nucleic acid or amino acid sequence that encodes the native sulfatase. In still another embodiment, the activity that is determined can be determining the enzyme's activity towards a particular substrate (e.g., the k_(cat) or K_(M) value (or ratio thereof) or the products of the reaction of the enzyme with the substrate).

In a further aspect, compositions comprising a sulfatase enzyme are also provided. In one embodiment, the compositions further comprise a pharmaceutically acceptable carrier.

In another embodiment, the compositions further comprise calcium.

In a further embodiment, the compositions do not contain a chelator. In another embodiment, the chelator that is not present is EDTA or EGTA. In still another embodiment, the compositions have a concentration of EDTA or EGTA that is less than or equal to 3 mM, 2 mM or 1 mM.

In another embodiment, the compositions provided herein do not contain sulfate ions, phosphate ions or both. In one embodiment, the compositions have a phosphate ion concentration of less than or equal to 5 mM or 2 mM. In another embodiment, the compositions have a sulfate ion concentration of less than or equal to 20 mM.

In yet another embodiment, the compositions provided have a sodium chloride concentration of less than 1 M. In a further embodiment, the compositions have a sodium chloride concentration of less than 0.5 M. In still a further embodiment, the sodium chloride concentration is less than 200 mM.

In still another embodiment, the compositions provided have a pH that is greater than 4.5. In a further embodiment, the pH of the compositions is less than 9. In one embodiment, the pH of the compositions is greater than 4.5 and less than 9. In another embodiment, the pH is in the range of 5-9. In yet another embodiment, the pH is in the range of 6-8. In still another embodiment, the pH is in the range of 5.5-6.5.

In another embodiment, the compositions provided further comprise acetate buffer.

In still another embodiment, the compositions provided further comprise at least one additional polysaccharide-degrading enzyme, such as a GAG-degrading enzyme. In one embodiment, the at least one additional GAG-degrading enzyme is heparinase I, heparinase II, heparinase III, 2-O sulfatase or Δ4,5 glycuronidase. In another embodiment, the at least one additional GAG-degrading enzyme is another sulfatase, glycosyl hydrolase, endoglucuronidase or lyase.

In a further aspect of the invention, compositions are provided that comprise a sulfatase enzyme and a solid support membrane, wherein the sulfatase is immobilized on the solid support membrane.

In still another aspect of the invention, nucleic acid molecules encoding the enzymes or polypeptides of the invention are provided. In one aspect, the nucleic acid is an isolated nucleic acid molecule selected from the group consisting of (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence set forth in SEQ ID NO: 1 or 3 (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code, and (c) complements of (a) or (b). In one embodiment, the complement is a full complement. In another embodiment the nucleic acid codes for a sulfatase. In yet another embodiment, the isolated nucleic acid molecule has a nucleotide sequence set forth in SEQ ID NO: 1 or a fragment thereof. In another embodiment, the isolated nucleic acid molecule has a nucleotide sequence set forth in SEQ ID NO: 3 or a fragment thereof. In one embodiment, the nucleic acid fragment encodes a polypeptide that has the amino acid sequence of SEQ ID NO: 2, 4, 17 or 18 or an amino acid sequence present therein. In another embodiment, such a polypeptide consists of at least 8, 9, 10, 11, 12, 15, 18, 20, 25, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450 or 550, etc. amino acids (up to the full-length of the enzyme amino acid sequence).

In another aspect of the invention, an expression vector comprising an isolated nucleic acid molecule as provided herein operably linked to a promoter is provided. In a further aspect of the invention, a host cell comprising the expression vector is provided. In still another aspect, compositions comprising the isolated nucleic acid molecule, vector or host cell are provided. In one embodiment, the compositions further comprise a pharmaceutically acceptable carrier.

In another aspect of the invention, methods of degrading monosaccharides or polysaccharides with the molecules provided herein are provided. In one aspect, the methods of degrading comprise contacting a glycosaminoglycan with a sulfatase or composition thereof in an amount effective to degrade the glycosaminoglycan. In one embodiment, the glycosaminoglycan is a HSGAG.

In another aspect of the invention, a degraded monosaccharide or polysaccharide (e.g., a degraded glycosaminoglycan, such as a degraded HSGAG) produced by a method provided herein is provided.

In a further aspect, a composition comprising the degraded monosaccharide or polysaccharide is provided. In another aspect, the composition further comprises a pharmaceutically acceptable carrier.

In still another aspect of the invention, methods of analyzing monosaccharides or polysaccharides with the molecules provided herein are provided. In one aspect, the methods of analyzing comprises contacting a glycosaminoglycan (e.g., a HSGAG) with a sulfatase or composition thereof in an amount effective to analyze the glycosaminoglycan. In one embodiment, the method is a method for identifying the presence of a particular glycosaminoglycan in a sample. In another embodiment, the method is a method for determining the purity of a glycosaminoglycan in a sample. In yet another embodiment, the method is a method for determining the composition of a glycosaminoglycan in a sample. In a further embodiment, the method is a method for determining the sequence of saccharide units in a glycosaminoglycan. In another embodiment, the method further comprises using an analytic technique, such as mass spectrometry, NMR spectroscopy, gel electrophoresis, capillary electrophoresis and/or HPLC.

In one embodiment, any of the methods provided can include contacting with at least one additional polysaccharide-degrading enzyme, such as a glycosaminoglycan-degrading enzyme. In another embodiment, the at least one additional glycosaminoglycan-degrading enzyme is used prior to, subsequent to or concurrently with a 6-O-sulfatase and/or N-sulfamidase. In a further embodiment, the at least one additional glycosaminoglycan-degrading enzyme is heparinase I, heparinse II, heparinase III, 2-O sulfatase or Δ4,5 glycuronidase. In yet a further embodiment, the at least one additional GAG-degrading enzyme is another sulfatase, glycosyl hydrolase, endoglucuronidase or lyase. In one embodiment, the at least one additional glycosaminoglycan-degrading enzyme is Δ4,5 glycuronidase. In another embodiment, the Δ4,5 glycuronidase is contacted with the glycosaminoglycan prior to the 6-O-sulfatase or N-sulfamidase. In a further embodiment, the glycosaminoglycan is contacted with a 2-O sulfatase and the contact with the 2-O sulfatase is prior to the contact with the Δ4,5 glycuronidase. In yet another embodiment, the glycosaminoglycan is contacted with a heparinase and the contact with the heparinase is prior to the contact with the 2-O sulfatase. In another embodiment, the glycosaminoglycan is contacted with 6-O sulfatase and with N-sulfamidase, wherein the glycosaminoglycan is contacted with the 6-O sulfatase prior to the N-sulfamidase. In a further embodiment, the glycosaminoglycan is 3-O desulfated prior to contact with 6-O sulfatase or N-sulfamidase.

In another aspect of the invention, methods of treatment with the compositions provided herein are provided. In one aspect, a method of inhibiting angiogenesis, comprising administering to a subject in need thereof an effective amount of a composition provided herein for inhibiting angiogenesis is provided. In another aspect, a method of treating cancer, comprising administering to a subject in need thereof an effective amount of a composition provided herein for treating cancer is provided. In still another aspect of the invention, a method of inhibiting metastasis, comprising administering to a subject in need thereof an effective amount of a composition provided herein for inhibiting metastasis is provided. In a further aspect, a method of treating a coagulation disorder in a subject, comprising administering a composition provided herein to a subject in need thereof an effective amount for treat the coagulation disorder is provided. In yet another aspect of the invention, a method of treating an inflammatory disorder, comprising administering a composition provided herein to a subject in need thereof an effective amount for treating the inflammatory disorder is provided.

In still another aspect of the invention, compositions and methods for treating the conditions provided herein include an additional therapeutic agent.

Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the flavobacterial 6-O-sulfatase (ORF B) coding sequence. The gene sequence (SEQ ID NO: 1) described as ORF B encodes a polypeptide with 548 amino acids (SEQ ID NO: 19) (545 amino acids starting at the initiating methionine (SEQ ID NO: 17)). The translated protein sequence is highlighted in bold. Numbering of amino acids is noted in parentheses and begins with initiating Met (position noted above corresponding ATG codon). The PFAM sulfatase motif CXPXRXXXXS/TG (SEQ ID NO: 5) is boxed. The predicted signal sequence is overscored with the peptide cleavage site represented by an arrow. Vicinal glutamines (Q) are shaded in gray. The Hind III restriction site is doubled underscored.

FIG. 2 provides the flavobacterial N-sulfamidase (ORF C) coding sequence. The gene sequence, described as ORF C (SEQ ID NO: 3), encodes a polypeptide with 507 amino acids (SEQ ID NO: 20) (500 amino acids starting at the initiating methionine (SEQ ID NO: 18)). The translated protein sequence is highlighted in bold. See FIG. 1 legend for details.

FIG. 3 shows a multiple sequence alignment for ORF B and putative functional assignment as a carbohydrate 6-O-desulfating enzyme. The cloned flavobacterial gene sequence corresponding to ORF B encodes a protein which is a member of a large sulfatase gene family identified by the PFAM sulfatase consensus sequence CXPXRXXXXS/TG (SEQ ID NO: 5). Shown in this representative alignment are the primary amino acid sequences of two bacterial enzymes to which the flavobacterial sequence (as set forth in SEQ ID NO: 17) exhibited the strongest homology: Bacteroides (abbr. Bacter.; SEQ ID NO: 6) and Prevotella sp. (abbr. Prev.; SEQ ID NO: 7) The sequence of the human galactosamine-6-O-sulfatase (Gal-6) is also shown for comparison (SEQ ID NO: 8). The putative functional assignment of the flavobacterial enzyme as a GAG 6-O-sulfatase is based on the functionality of the two bacterial enzymes (especially the Prevotella sp. mucin desulfating enzyme which specifically hydrolyzes the N-acetylglucosamine 6-O-sulfate). Sequence alignment was generated using CLUSTALW (EMBL, Heidelberg, Germany). A consensus sequence is also shown (SEQ ID NO: 21).

FIG. 4 shows a multiple sequence alignment for ORF C and putative functional assignment as a heparan/heparin N-sulfamidase. The cloned flavobacterial gene sequence corresponding to ORF C likewise encodes a protein which is a member of a large sulfatase gene family. As was the case for the 6-O-sulfatase, the functional assignment of the flavobacterial enzyme (as set forth in SEQ ID NO: 20) as a GAG N-sulfamidase is inferred from the putative functionality of the two bacterial enzymes to which it shows the strongest sequence homology: Pirellula sp. (abbr. Pirell.; SEQ ID NO: 9) and Bacteroides (abbr. Bacter.; SEQ ID NO: 22). Also included in this alignment is the primary amino acid sequence of the human lysosomal enzyme, heparin-N-sulfamidase (SEQ ID NO: 10). A consensus sequence is also shown (SEQ ID NO: 23).

FIG. 5 demonstrates the substrate specificity of recombinant 6-O-sulfatase. FIG. 5A shows the specificity of the 6-O-sulfatase as a heparin/heparan desulfating enzyme. Desulfation of 4MUGal-6S (i), 4MUGalNAc,6S (ii), or 4MUGlcNAc,6S (iii and iv) by the recombinant 6-O-sulfatase was followed by capillary electrophoresis. Exclusive desulfation of 4MUGlcNAc,6S by 6-O-sulfatase is evidenced by a singular disappearance of absorbance at 315 nm in electrophoretagram (iii) (that normally appears at approximately 4 minutes). Minus enzyme control is shown in (iv). Electrophoretagrams are offset for illustrative purposes. FIG. 5B shows results from the timecourse analyses of recombinant 6-O-sulfatase activity using three different, 6-O-sulfated monosaccharide substrates (each at 2.5 μM). GlcNAc,6S (●), GlcNS,6S (◯), 4MUGlcNAc,6S (▴). Inset: time course out to 30 minutes.

FIG. 6 illustrates the obligatory substrate-product relationship of the 6-O-sulfatase and N-sulfamidase. Desulfation of the disulfated monosaccharide H_(NS,6S) by the two enzymes was followed by electrospray mass spectrometry (ESI-MS). Panel i): substrate only shown as the sodium adduct of a single ion species (M-1) with molecular mass of 360.8 Da; ii) desulfation of disulfated monosaccharide by 6-O-sulfatase, resulting in the monosulfated product (258.1 Da); iii) inability of N-sulfamidase to hydrolyze the original disulfated monosaccharide (compare with i); iv) co-treatment of the disulfated substrate with both enzymes showing the disappearance of all sulfated monosaccharides and demonstrating 6-O-desulfation by the 6-O-sulfatase prior to sulfate hydrolysis at the 2N position by the N-sulfamidase. Internal standard (458.1 Da) used in mass calibration is noted by an asterisk.

FIG. 7 provides sulfatase reaction conditions. FIG. 7A demonstrates the inhibition by NaCl. Results are normalized to 0.0 M NaCl added (which is shown as 100%). 6-O-sulfatase (●); N-sulfamidase (◯). FIG. 7B demonstrates the inhibition by sulfate (●) or phosphate (◯). Results for 6-O-sulfatase are represented by a solid line; results for N-sulfamidase are represented by a dashed line. Results are normalized to 0 sulfate or phosphate added. Log scale for inhibitor concentration, [I]. FIGS. 7C and 7D show the pH profile of sulfatase activity for 6-O-sulfatase and N-sulfamidase, respectively. Relative activities (% desulfation) were measured over a pH range of 4-8 using three buffers: sodium acetate (●), MES (◯) and MOPS (▴).

FIG. 8 demonstrates the effect of divalent metals on sulfatase activities. FIGS. 8A and 8B illustrates the calcium specific activation of 6-O-sulfatase activity and inhibition by EDTA (FIG. 8A). The calcium-specific requirement for N-sulfamidase activity and inhibition by EDTA is shown in FIG. 8B. In both cases, the divalent metal effect was not observed when calcium was replaced by either Mg⁺² or Mn⁺² (at either 1 mM or 5 mM concentrations). Open bars (no divalent metals added); black bars (1 mM EDTA added); light gray bars (1 mM divalent metal); stippled gray bars (5 mM divalent metal). FIGS. 8C and 8D illustrate the effect of calcium on steady-state kinetics. Enzyme kinetics were measured for the 6-O-sulfatase (FIG. 8C) and N-sulfamidase (FIG. 8D) as described in Materials and Methods at varying concentrations of Ca⁺² or in the presence of 1 mM EDTA. Substrate saturation plots were fitted to pseudo first-order Michaelis-Menten kinetics by non-linear regression analyses. 0.5 mM Ca⁺² (●), 1 mM Ca⁺² (◯), 5 mM Ca⁺² (▪), 1 mM EDTA (Δ). The EDTA result (showing a lack of activity) is omitted in FIG. 8D.

FIG. 9 demonstrates the sequential degradation of a HSGAG tetrasaccharide using recombinantly expressed flavobacterial enzymes. The ability of the 6-O-sulfatase to hydrolyze the non-reducing end of an oligosaccharide is demonstrated in the context of exo-sequencing the heparin derived tetrasaccharides ΔU_(2S)H_(NS,6S)I_(2S)H_(NS,6S) and structurally related ΔUH_(NS,6S)I_(2S)H_(NS,6S) lacking a 2-O-sulfate at the internal iduronic acid position. Sequential treatment of the HSGAG tetrasaccharide was physically assessed after each enzyme step (FIG. 9A-9E) by MALDI-MS. Masses listed in each panel represent either peptide alone (˜3216 Da) or oligosaccharide-peptide complex. The net mass of the oligosaccharide is listed in parentheses. FIG. 9A provides results from the addition of the 2-O-sulfatase; FIG. 9B provides results from the subsequent addition of the Δ4,5 glycuronidase; FIG. 9C provides results from the subsequent addition of the 6-O-sulfatase (note loss of a sulfate represented by a shift in net molecular mass from ˜915 to ˜835 Da); FIG. 9D provides results from the addition of N-sulfamidase directly after the Δ4,5 glycuronidase step (note the lack of any desulfation); FIG. 9E provides results from the addition of the N-sulfamidase subsequent to the 6-O-sulfatase. The results in FIG. 9E are equivocal inasmuch as a double desulfated species with a net molecular mass of ˜755 Da was not clearly detected in this experiment.

FIG. 10 demonstrates the sequential degradation of a HSGAG hexasaccharide. As in FIG. 9, the use of the flavobacterial exo-enzymes for sequentially degrading a HSGAG oligosaccharide is presented. In this experiment, the ability of the N-sulfamidase to desulfate the non-reducing end of an oligosaccharide is also demonstrated. The structurally-defined, heparin derived hexasaccharide ΔUH_(NS)IH_(NS)IH_(NS) was treated with Δ4,5 glycuronidase alone or with Δ4,5 glycuronidase followed by the N-sulfamidase. Resultant oligosaccharide products were fluorescently labeled at the reducing end with APTS by reductive amination as described in Materials and Methods. Oligosaccharides were resolved by capillary electrophoresis and detected by laser-induced fluorescence (CE-LIF). The N-desulfated pentasaccharide H_(NH2)IH_(NS)IH_(NS) is observed as a unique peak appearing at approximately 7.6 minutes (peak Z).

FIG. 11 shows a structure-oriented multiple sequence alignment of cloned flavobacterial sulfatases. The sequence alignment of ORF B (6-O-sulfatase) (as set forth in SEQ ID NO: 17) and ORF C(N-sulfamidase) (as set forth in SEQ ID NO: 18) with the primary sequences of three sulfatases (bacterial arylsulfatase from Pseudomonas aeruginosa (PARSA) (SEQ ID NO: 26), human arylsulfatase A (Human ARS A) (SEQ ID NO: 25), and human arylsulfatase B (Human ARS B) (SEQ ID NO: 24), which is actually a N-acetylgalactosamine-4-sulfatase) is shown. Select residues known to comprise the enzyme active site are boxed. The position of the catalytic cysteine modification is noted by an asterisk. A consensus sequence is also shown (SEQ ID NO: 27).

DETAILED DESCRIPTION OF THE INVENTION

Previously the cloning, characterization, and recombinant expression in Escherichia coli of the Flavobacterium heparinum (a.k.a. Pedobacter heparinus) 2-O-sulfatase was reported. Two additional sulfatases have been cloned, a 6-O-sulfatase and a N-sulfamidase from the same microorganism. These two enzymes were expressed in E. coli in a soluble, active form and their functionality as HSGAG sulfatases was confirmed and their respective kinetic and biochemical properties determined.

The two sulfatase genes described were identified during the process of screening a genomic library with hybridization probes directed toward the flavobacterial 2-O-sulfatase. Two overlapping phagemid clones identified during this process were expanded by chromosomal walking and restriction mapping. Sequence analyses of this genomic region revealed two sizeable open reading frames of 1647 and 1524 base pairs (described hereafter as ORF B and ORF C, respectively). The two gene sequences putatively encode proteins of 545 and 500 amino acids in length (starting at the initiating Met). Neither sequence possessed an obvious Shine-Dalgarno ribosomal binding site within 10 nucleotides of the initiating ATG codon. A closer examination of their respective sequences at the protein level noted several important features. Both flavobacterial ORFs possess a N-terminal hydrophobic signal peptide and corresponding cleavage site sequence predicted by the Von Heijne method for gram-negative bacteria (Nielsen et al., 1997 Protein Eng 10, 1-6). Both genes encode basic protein sequences of comparable amino acid composition (by mol percent). Of the two proteins, the ORF B gene product possesses a slightly higher theoretical pI (8.6 vs. 8.0) relative to ORF C. A BLASTP sequence homology search of the two flavobacterial genes against the protein database unambiguously identified both gene products as members of a large sulfatase family (BLASTP, NLM; Bethesda, Md.). Both protein sequences possess the signature PFAM sulfatase motif C/SXPXRXXXXS/TG (SEQ ID NO: 5) as well as the highly conserved sequence LTG (at the +9 through +11 positions relative to this motif). As is the case for many other sulfatases that comprise this large enzyme family, this sulfatase domain is likewise located in the N-terminal region of the encoded polypeptide.

The data presented here demonstrate that both the 6-O-sulfatase and the N-sulfamidase are exolytic enzymes. In addition, in the studies performed, it was found that the 6-O-sulfatase was strongly activated by calcium and inhibited by sulfate and phosphate, while the N-sulfamidase requires calcium but was not apparently inhibited by either sulfate or phosphate. The 6-O-sulfatase was shown to act on either N-sulfated or N-acetylated 6-O-sulfated glucosamines, while being completely inhibited by 3-O-sulfation or unsubstituted amines on the same pyranose ring. The N-sulfamidase was shown to act solely on N-sulfated glucosamines, while being completely inhibited by 3-O or 6-O sulfation. Both enzymes were completely inactive when a glycosidically linked uronic acid was present at the non-reducing C4 position. Taken together with the reported substrate specificities for the previously characterized F. Heparinum 2-O-sulfatase and unsaturated glucuronyl hydrolase, the in vitro exolytic sequence for the heparin and heparan sulfate degradation pathway of F. heparinum was defined. In addition, these enzymes can be applied in tandem toward the exo-sequencing of heparin-derived oligosaccharides.

The invention provides, in part, 6-O-sulfatase and N-sulfamidase enzymes as well as compositions thereof and methods of their use. Also provided are polypeptides comprising the amino acid sequence of either enzyme as well as nucleic acids that encode them. A polypeptide that comprises the amino acid sequence of 6-O-sulfatase, therefore, can comprise the amino acid sequence provided in FIG. 1 starting at the initiating methionine (SEQ ID NO: 17) or the amino acid sequence of the mature 6-O-sulfatase polypeptide (SEQ ID NO: 2) (i.e., SEQ ID NO: 17 without the signal sequence). Likewise, a polypeptide that comprises the amino acid sequence of N-sulfamidase can comprise the amino acid sequence provided in FIG. 2 starting at the initiating methionine (SEQ ID NO: 18) or the amino acid sequence of the mature N-sulfamidase polypeptide (SEQ ID NO: 4) (i.e., SEQ ID NO: 18 without the signal sequence). Fragments of these polypeptides and compositions that contain them are also provided. In some embodiments the fragments are at least 8, 9, 10, 11, 12, 15, 18, 20, 25, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 550, etc. up to the full-length of SEQ ID NO: 2, 4, 17 or 18 minus 1. In some embodiments the cysteine of the cysteine active site of the 6-O-sulfatase or N-sulfamidase is present in its modified form (FGly) in the enzyme or polypeptide provided.

Polypeptides (or enzymes) can be isolated from biological samples, and can also be expressed recombinantly in a variety of prokaryotic and eukaryotic expression systems, such as those described below, by constructing an expression vector appropriate to the expression system, introducing the expression vector into the expression system, and isolating the recombinantly expressed protein. Polypeptides can also be synthesized chemically using well-established methods of peptide synthesis.

The polypeptides (or enzymes) provided herein are in some embodiments isolated or substantially pure. As used herein, “isolated” means the polypeptide or enzyme is separated from its native environment and present in sufficient quantity to permit its identification or use. This means, for example: (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis. Isolated proteins or polypeptides may be, but need not be, substantially pure. Because an isolated polypeptide or enzyme may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the polypeptide or enzyme may comprise only a small percentage by weight of the preparation. The polypeptide or enzyme is nonetheless isolated in that it has been separated from the substances with which it may be associated in living systems, i.e., isolated from other proteins.

As used herein, the term “substantially pure” means that the proteins or polypeptides are essentially free of other substances to an extent practical and appropriate for their intended use. In particular, the proteins are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, protein sequencing, or producing pharmaceutical preparations. As used herein, a “substantially pure 6-O-sulfatase or N-sulfamidase” is a preparation of 6-O-sulfatase or N-sulfamidase, respectively, which has been isolated or synthesized and which is greater than about 90% free of contaminants. Preferably, the material is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even greater than 99% free of contaminants. The degree of purity may be assessed by means known in the art. One method for assessing the purity of the material may be accomplished through the use of specific activity assays.

Recombinant 6-O-sulfatase or N-sulfamidase enzymes are also provided. As used herein, a “recombinant 6-O-sulfatase or N-sulfamidase” is a 6-O-sulfatase or N-sulfamidase that has been produced through human manipulation of a nucleic acid that encodes the enzyme. The human manipulation usually involves joining a nucleic acid that encodes the 6-O-sulfatase or N-sulfamidase to the genetic material of a different organism and, generally, a different species. “Recombinant” is a term of art that is readily known to one of skill, and techniques for the recombinant expression of 6-O-sulfatase or N-sulfamidase are readily available to those of skill in the art and include those described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998). Other techniques for recombinant expression including examples of expression systems are described further below.

Recombinant technology can also be used to produce modified versions of 6-O-sulfatase or N-sulfamidase. As used herein, “modified” refers to any alteration of the enzyme as compared to the native enzyme (i.e., as it would be found in nature). The modified 6-O-sulfatase, in some embodiments, can comprise an amino acid sequence set forth in SEQ ID NO: 2, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is the residue at position 39, 40, 80, 83, 127, 129, 206, 277, 386 or 503. The modified 6-O-sulfatase, in other embodiments, can comprise an amino acid sequence set forth in SEQ ID NO: 2, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is the residue at position 40, 41, 80, 84, 128, 130, 206, 277, 387 or 504. In another embodiment, the amino acid that has been substituted or deleted is at position 207. In still another embodiment, the amino acid that has been substituted or deleted is at position 276. In other embodiments, the modified 6-O-sulfatase comprises an amino acid sequence set forth in SEQ ID NO: 2, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is not the residue at position 40, 41, 80, 84, 128, 130, 206, 277, 387 or 504. In another embodiment, the amino acid that has not been substituted or deleted is at position 207. In still another embodiment, the amino acid that has not been substituted or deleted is at position 276. In some of these embodiments the cysteine of the cysteine active site is present in its modified form (FGly).

In other embodiments, it is the N-sulfamidase that is modified. For example, the modified N-sulfamidase can comprise an amino acid sequence set forth in SEQ ID NO: 4, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is the residue at position 39, 80, 83, 128, 175 or 245. In another embodiment, the modified N-sulfamidase can comprise an amino acid sequence set forth in SEQ ID NO: 4, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is the residue at position 40, 80, 84, 129, 176 or 246. In a further embodiment, the modified N-sulfamidase can comprise an amino acid sequence set forth in SEQ ID NO: 4, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is not the residue at position 39, 80, 83, 128, 175 or 245. In still a further embodiment, the modified N-sulfamidase can comprise an amino acid sequence set forth in SEQ ID NO: 4, wherein at least one amino acid residue has been substituted or deleted, and wherein the at least one amino acid residue that has been substituted or deleted is not the residue at position 40, 80, 84, 129, 176 or 246. In some of these embodiments the cysteine of the cysteine active site is present in its modified form (FGly).

Based on the understanding of the important residues involved, functional variants can be produced. As used herein, a “functional variant” of a 6-O-sulfatase and N-sulfamidase polypeptide is a polypeptide which contains one or more modifications to the primary amino acid sequence of the 6-O-sulfatase and N-sulfamidase polypeptide, respectively. The polypeptide can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50 or more amino acid modifications. These modifications are intended to encompass modifications that result in a 6-O-sulfatase or N-sulfamidase with altered activity relative to the native 6-O-sulfatase or N-sulfamidase but also include modifications that do not result in altered activity relative to the native enzyme. The term “native” as used herein refers to the 6-O-sulfatase or N-sulfamidase as it would be found in nature. Modifications which create a 6-O-sulfatase or N-sulfamidase polypeptide functional variant are typically made to the nucleic acid which encodes the 6-O-sulfatase or N-sulfamidase polypeptide, and can include deletions, point mutations, truncations, amino acid substitutions and addition of amino acids or non-amino acid moieties to, for example: 1) enhance a property of a 6-O-sulfatase or N-sulfamidase polypeptide, such as protein stability in an expression system or the stability of protein-protein binding; 2) provide a novel activity or property to a 6-O-sulfatase or N-sulfamidase polypeptide, such as addition of a detectable moiety; or 3) to provide equivalent or better interaction with other molecules (e.g., heparin). Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the 6-O-sulfatase or N-sulfamidase amino acid sequences. One of skill in the art will be familiar with methods for predicting the effect on protein conformation of a change in protein sequence, and can thus “design” a functional variant 6-O-sulfatase or N-sulfamidase polypeptide according to known methods. One example of such a method is described by Dahiyat and Mayo in Science 278:82-87, 1997, whereby proteins can be designed de novo. The method can be applied to a known protein to vary only a portion of the polypeptide sequence. By applying the computational methods of Dahiyat and Mayo, specific variants of a polypeptide can be proposed and tested to determine whether the variant retains a desired conformation.

Functional variants can include 6-O-sulfatase or N-sulfamidase polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Similarly, certain amino acids can be changed to enhance expression of a 6-O-sulfatase or N-sulfamidase polypeptide by eliminating proteolysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present). Functional variants, therefore, can also include variant 6-O-sulfatase or N-sulfamidase that maintain the same enzymatic function as the native 6-O-sulfatase or N-sulfamidase but include some modification to the amino acid sequence that does not alter native enzyme activity. These modifications include conservative amino acid substitutions as well as non-conservative amino acid substitutions that are remote from the binding and catalytic sites of the enzyme.

Mutations of a nucleic acid which encode a 6-O-sulfatase or N-sulfamidase polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such as hairpins or loops, which can be deleterious to expression of the variant polypeptide.

Mutations can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. Variant polypeptides are then expressed and tested for one or more activities to determine which mutation provides a variant polypeptide with the desired properties. Further mutations can be made to variants (or to non-variant 6-O-sulfatase or N-sulfamidase polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli, are well known to those of ordinary skill in the art. Still other mutations can be made to the noncoding sequences of a 6-O-sulfatase or N-sulfamidase gene or cDNA clone to enhance expression of the polypeptide.

In the description herein, reference is made to the amino acid residues and residue positions of native 6-O-sulfatase or N-sulfamidase (SEQ ID NO: 17 and 18, respectively). In particular, residues and residue positions will be referred to as “corresponding to” a particular residue or residue position of 6-O-sulfatase or N-sulfamidase. As will be obvious to one of ordinary skill in the art, these positions are relative and, therefore, insertions or deletions of one or more residues would have the effect of altering the numbering of downstream residues. In particular, N-terminal insertions or deletions would alter the numbering of all subsequent residues. Therefore, as used herein, a residue in a modified enzyme will be referred to as “corresponding to” a residue of the native enzyme if, using standard sequence comparison programs, they would be aligned. Many such sequence alignment programs are now available to one of ordinary skill in the art and their use in sequence comparisons has become standard (e.g., “LALIGN” available via the Internet at phaedra.crbm.cnrs-mop.fr/fasta/lalign-query.html). As used herein, this convention of referring to the positions of residues of the recombinant modified heparinases by their corresponding 6-O-sulfatase or N-sulfamidase residues shall extend not only to embodiments including N-terminal insertions or deletions but also to internal insertions or deletions (e.g., insertions or deletions in “loop” regions).

One type of amino acid substitution is referred to as a “conservative substitution.” As used herein, a “conservative amino acid substitution” or “conservative substitution” refers to an amino acid substitution in which the substituted amino acid residue is of similar charge as the replaced residue and is of similar or smaller size than the replaced residue. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) the small non-polar amino acids, A, M, I, L, and V; (b) the small polar amino acids, G, S, T and C; (c) the amido amino acids, Q and N; (d) the aromatic amino acids, F, Y and W; (e) the basic amino acids, K, R and H; and (f) the acidic amino acids, E and D. Substitutions which are charge neutral and which replace a residue with a smaller residue may also be considered “conservative substitutions” even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine). The term “conservative amino acid substitution” also refers to the use of amino acid analogs.

Methods for making amino acid substitutions, additions or deletions are well known in the art. The terms “conservative substitution”, “non-conservative substitutions”, “non-polar amino acids”, “polar amino acids”, and “acidic amino acids” are all used consistently with the prior art terminology. Each of these terms is well-known in the art and has been extensively described in numerous publications, including standard biochemistry text books, such as “Biochemistry” by Geoffrey Zubay, Addison-Wesley Publishing Co., 1986 edition, which describes conservative and non-conservative substitutions, and properties of amino acids which lead to their definition as polar, non-polar or acidic.

One skilled in the art will appreciate that the effect can be evaluated by routine screening assays, preferably the biological assays described herein. Modifications of peptide properties including thermal stability, enzymatic activity, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers are assayed by methods well known to the ordinarily skilled artisan. For additional detailed description of protein chemistry and structure, see Schulz, G. E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1979, and Creighton, T. E., Proteins: Structure and Molecular Principles, W.H. Freeman & Co., San Francisco, 1984.

Additionally, some of the amino acid substitutions are non-conservative substitutions. In certain embodiments where the substitution is remote from the active or binding sites, the non-conservative substitutions are easily tolerated provided that they preserve a tertiary structure characteristic of, or similar to, native 6-O-sulfatase or N-sulfamidase, thereby preserving the active and binding sites. Non-conservative substitutions, such as between, rather than within, the above groups (or two other amino acid groups not shown above), which will differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

The enzymes, can be recombinantly produced using a vector including a coding sequence operably joined to one or more regulatory sequences. As used herein, a coding sequence and regulatory sequences are said to be “operably joined” when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein the coding sequences are operably joined to regulatory sequences. Two DNA sequences are said to be operably joined if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.

The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5′ non-transcribing and 5′ non-translating sequences involved with initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5′ non-transcribing regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Promoters may be constitutive or inducible. Regulatory sequences may also include enhancer sequences or upstream activator sequences, as desired.

As used herein, a “vector” may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids and phagemids. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium, or just a single time per host as the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques. Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.

For prokaryotic systems, plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host may be used. Examples of suitable plasmid vectors include pBR322, pUC18, pUC19 and the like; suitable phage or bacteriophage vectors include λgt10, λgt11 and the like; and suitable virus vectors include pMAM-neo, pKRC and the like. Preferably, the selected vector of the present invention has the capacity to autonomously replicate in the selected host cell. Useful prokaryotic hosts include bacteria, in addition to E. coli, Flavobacterium heparinum, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like.

To express the enzymes of the invention in a prokaryotic cell, it is desirable to operably join the nucleic acid sequence to a functional prokaryotic promoter. Such promoter may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage λ, the bla promoter of the β-lactamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage λ (P_(L) and P_(R)), the trp, recA, lacZ, lacI, and gal promoters of E. coli, the α-amylase (Ulmanen et al., J. Bacteriol. 162:176-182 (1985)) and the ζ-28-specific promoters of B. subtilis (Gilman et al., Gene sequence 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward et al., Mol. Gen. Genet. 203:468-478 (1986)).

Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo (Biochimie 68:505-516 (1986)); and Gottesman (Ann. Rev. Genet. 18:415-442 (1984)).

Proper expression in a prokaryotic cell also requires the presence of a ribosome binding site upstream of the encoding sequence. Such ribosome binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35:365-404 (1981)).

Because prokaryotic cells may not produce the enzymes of the invention with glycosylation, expression of the enzymes of the invention in eukaryotic hosts is useful when glycosylation is desired. Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, and mammalian cells, either in vivo or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin, such as the hybridoma SP2/0-AG14 or the myeloma P3x63Sg8, and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332 that may provide better capacities for correct post-translational processing. Embryonic cells and mature cells of a transplantable organ also are useful according to some aspects of the invention.

In addition, plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences.

Another preferred host is an insect cell, for example in Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used (Rubin, Science 240:1453-1459 (1988)). Alternatively, baculovirus vectors can be engineered to express large amounts of the enzymes of the invention in insect cells (Jasny, Science 238:1653 (1987); Miller et al., In: Genetic Engineering (1986), Setlow, J. K., et al., eds., Plenum, Vol. 8, pp. 277-297).

Any of a series of yeast gene sequence expression systems which incorporate promoter and termination elements from the genes coding for glycolytic enzymes and which are produced in large quantities when the yeast are grown in media rich in glucose may also be utilized. Known glycolytic gene sequences can also provide very efficient transcriptional control signals. Yeast provide substantial advantages in that they can also carry out post-translational peptide modifications. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number plasmids which can be utilized for production of the desired proteins in yeast. Yeast recognize leader sequences on cloned mammalian gene sequence products and secrete peptides bearing leader sequences (i.e., pre-peptides).

A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, and the like, may be employed. Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated. Of interest are regulatory signals that are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or which are subject to chemical (such as metabolite) regulation.

As discussed above, expression of the enzymes of the invention in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, Cell 31:355-365 (1982)); the SV40 early promoter (Benoist et al., Nature (London) 290:304-310 (1981)); the yeast gal4 gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the enzyme of the invention does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in the formation of a fusion protein (if the AUG codon is in the same reading frame as the enzyme of the invention coding sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the enzyme of the invention coding sequence).

In one embodiment, a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may, for example, provide for prototrophy to an auxotrophic host or may confer biocide resistance to, e.g., antibiotics, heavy metals, or the like. The selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of 6-O-sulfatase and N-sulfamidase mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama, Molec. Cell. Biol. 3:280 (1983).

In another embodiment, the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species. Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColE1, pSC101, pACYC 184, and πVX). Such plasmids are, for example, disclosed by Sambrook, et al. (Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989)). Bacillus plasmids include pC194, pC221, pT127, and the like. Such plasmids are disclosed by Gryczan (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include pIJ101 (Kendall et al., J. Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages such as φC31 (Chater et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki (Jpn. J. Bacteriol. 33:729-742 (1978)).

Preferred eukaryotic plasmids include, for example, BPV, EBV, SV40, 2-micron circle, and the like, or their derivatives. Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274 (1982); Broach, In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, Cell 28:203-204 (1982); Bollon et al., J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608 (1980)). Other preferred eukaryotic vectors are viral vectors. For example, and not by way of limitation, the pox virus, herpes virus, adenovirus and various retroviruses may be employed. The viral vectors may include either DNA or RNA viruses to cause expression of the insert DNA or insert RNA.

Once the vector or DNA sequence containing the construct(s) has been prepared for expression, the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, and the like. Additionally, DNA or RNA encoding the polypeptides of the invention may be directly injected into cells or may be impelled through cell membranes after being adhered to microparticles. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of polypeptide (or enzyme). This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like).

One of skill in the art may also substitute appropriate codons to produce the desired amino acid substitutions by standard site-directed mutagenesis techniques. One may also use any sequence which differs from the nucleic acid equivalents of the nucleic acids that encode the polypeptides of the invention, e.g., SEQ ID NO: 1, SEQ ID NO: 3 or fragments thereof, only due to the degeneracy of the genetic code as the starting point for site directed mutagenesis. The mutated nucleic acid sequence may then be ligated into an appropriate expression vector and expressed in a host such as E. coli.

The invention, therefore, also provides the isolated nucleic acid molecules that code for the 6-O-sulfatase enzymes, N-sulfamidase enzymes or polypeptides as described herein. The term “isolated nucleic acid”, as used herein, means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5′ and 3′ restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.

According to the invention, isolated nucleic acid molecules that code for a 6-O-sulfatase or N-sulfamidase enzyme or polypeptide include: (a) nucleic acid molecules which hybridize under stringent conditions to a nucleotide sequence set forth in SEQ ID NO: 1 or 3, which code for a 6-O-sulfatase or N-sulfamidase (enzyme or polypeptide), respectively; (b) deletions, additions and substitutions of (a) which code for a 6-O-sulfatase or N-sulfamidase (enzyme or polypeptide), respectively, or parts thereof; (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, and (d) complements of (a), (b) or (c) (e.g., full complements). The isolated nucleic acid molecules include, in some embodiments, isolated nucleic acid molecules that code for a 6-O-sulfatase or N-sulfamidase (enzyme or polypeptide) which has an amino acid sequence set forth as SEQ ID NOs: 2 or 4, respectively.

The invention also includes degenerate nucleic acids which include alternative codons to those present in the native materials. For example, serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is equivalent for the purposes of encoding a serine residue. Thus, it will be apparent to one of ordinary skill in the art that any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue into an elongating 6-O-sulfatase or N-sulfamidase polypeptide. Similarly, nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons). Other amino acid residues may be encoded similarly by multiple nucleotide sequences. Thus, the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.

The isolated nucleic acid molecules of the invention are also intended to encompass homologs and alleles which can be identified by conventional techniques. Identification of human and other organism homologs of 6-O-sulfatase or N-sulfamidase polypeptides will be familiar to those of skill in the art. In general, nucleic acid hybridization is a suitable method for identification of homologous sequences of another species, which correspond to a known sequence. Standard nucleic acid hybridization procedures can be used to identify related nucleic acid sequences of selected percent identity. For example, one can construct a library of cDNAs reverse transcribed from the mRNA of a sample and use the nucleic acids that encode a 6-O-sulfatase or N-sulfamidase identified therein to screen the library for related nucleotide sequences. The screening preferably is performed using high-stringency conditions to identify those sequences that are closely related by sequence identity. Nucleic acids so identified can be translated into polypeptides and the polypeptides can be tested for activity.

The term “stringent conditions” as used herein refers to parameters with which the art is familiar. Such parameters include salt, temperature, length of the probe, etc. The amount of resulting base mismatch upon hybridization can range from near 0% (“high stringency”) to about 30% (“low stringency”). Nucleic acid hybridization parameters may be found in references that compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. One example of high-stringency conditions is hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH2PO4 (pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid. After hybridization, a membrane upon which the nucleic acid is transferred is washed, for example, in 2×SSC at room temperature and then at 0.1-0.5×SSC/0.1×SDS at temperatures up to 68° C.

The skilled artisan also is familiar with the methodology for screening cells for expression of such molecules, which then are routinely isolated, followed by isolation of the pertinent nucleic acid. Thus, homologs and alleles of the 6-O-sulfatase and N-sulfamidase of the invention, as well as nucleic acids encoding the same, may be obtained routinely, and the invention is not intended to be limited to the specific sequences disclosed. It will be understood that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of homologs and alleles of the 6-O-sulfatase and N-sulfamidase nucleic acids of the invention. The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid molecule and sequencing.

In general, homologs and alleles typically will share at least 90% nucleotide identity and/or at least 95% amino acid identity to the sequences of the 6-O-sulfatase nucleic acids or N-sulfamidase nucleic acids or polypeptides, respectively. In some instances at least 95% nucleotide identity and/or at least 97% amino acid identity is shared. In other instances at least 97% nucleotide identity and/or at least 98% amino acid identity is shared, while in other instances at least 99% nucleotide identity and/or at least 99% amino acid identity is shared. In still other instances at least 99.5% nucleotide identity and/or at least 99.5% amino acid identity is shared. The homology can be calculated using various, publicly available software tools developed by NCBI (Bethesda, Md.) that can be obtained through the internet. Exemplary tools include the BLAST system available from the website of the National Center for Biotechnology Information (NCBI) at the National Institutes of Health. Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis can be obtained using the MacVector sequence analysis software (Oxford Molecular Group). Watson-Crick complements of the foregoing nucleic acids also are embraced by the invention.

In screening for the 6-O-sulfatase or N-sulfamidase related genes, such as homologs and alleles of the 6-O-sulfatase and N-sulfamidase, a Southern blot may be performed using the foregoing conditions, together with a radioactive probe. After washing the membrane to which the DNA is finally transferred, the membrane can be placed against X-ray film or a phosphoimager plate to detect the radioactive signal.

The present invention also provides for the use of the 6-O-sulfatase and N-sulfamidase molecules provided as enzymatic tools. The molecules include those with the native sequence as well as molecules that are fragments or functional variants of the native. A “native 6-O-sulfatase or N-sulfamidase specific activity” is the measure of enzymatic activity of native 6-O-sulfatase or N-sulfamidase obtained from cell lysates of F. heparinum. Therefore, based on the disclosure provided herein, those of ordinary skill in the art will be able to identify other 6-O-sulfatase or N-sulfamidase enzymes having altered enzymatic activity with respect to the native 6-O-sulfatase or N-sulfamidase, such as functional variants. The term “specific activity” as used herein refers to the enzymatic activity of a preparation of 6-O-sulfatase or N-sulfamidase.

The methods that may be used to test the specific activity of the 6-O-sulfatase and N-sulfamidase of the present invention are known in the art, e.g., those described in the Examples or as identified by the alignment provided in FIG. 11. These methods may also be used to assess the function of variants and functionally active fragments of 6-O-sulfatase or N-sulfamidase. For example, the k_(cat) value may be determined using any enzymatic activity assay to assess the activity of a 6-O-sulfatase or N-sulfamidase enzyme. Several such assays are well-known in the art. For instance, an assay for measuring k_(cat) is described in Ernst, S. E., Venkataraman, G., Winkler, S., Godavarti, R., Langer, R., Cooney, C. and Sasisekharan. R. (1996) Biochem. J. 315, 589-597. Therefore, based on the disclosure provided herein, those of ordinary skill in the art will be able to identify other 6-O-sulfatase or N-sulfamidase molecules having enzymatic activity that is similar to or altered in comparison with the native 6-O-sulfatase or N-sulfamidase molecule, such as 6-O-sulfatase or N-sulfamidase functional variants.

The activity of an enzyme can also be assessed according to its “product profile” (i.e., a characterization of the enzymatic products that result from contact of the enzyme with a monosaccharide or polysaccharide or a sample containing the monosaccharide or polysaccharide). The product profile may be determined by any method known in the art for examining the type or quantity of degradation products produced by a 6-O-sulfatase or N-sulfamidase alone or in combination with other enzymes. One preferred method for determining the type and quantity of product is described in Rhomberg, A. J. et al., PNAS, v. 95, p. 4176-4181, (April 1998), which is hereby incorporated in its entirety by reference. The method disclosed in the Rhomberg reference utilizes a combination of mass spectrometry and capillary electrophoretic techniques to identify the enzymatic products produced by heparinase. The Rhomberg study utilizes heparinase to degrade HSGAGs to produce HSGAG oligosaccharides. MALDI (Matrix-Assisted Laser Desorption Ionization) mass spectrometry can be used for the identification and semiquantitative measurement of substrates, enzymes, and end products in the enzymatic reaction. The capillary electrophoresis technique separates the products to resolve even small differences amongst the products and is applied in combination with mass spectrometry to quantitate the products produced. Capillary electrophoresis may even resolve the difference between a disaccharide and its semicarbazone derivative.

Other methods for assessing the product profile may also be utilized. For instance, other methods include methods which rely on parameters such as viscosity (Jandik, K. A., Gu, K. and Linhardt, R. J., (1994), Glycobiology, 4:284-296) or total UV absorbance (Ernst, S. et al., (1996), Biochem. J., 315:589-597) or mass spectrometry or capillary electrophoresis alone.

The 6-O-sulfatase and N-sulfamidase of the invention are useful as tools for degrading and/or analyzing monosaccharides (e.g., a monosaccharide of a GAG) or polysaccharides, such as sulfated polysaccharides (e.g., GAGs). The invention, therefore, includes a variety of in vitro, in vivo and ex vivo methods in which it is useful to degrade or analyze monosaccharides or polysaccharides or to determine whether or not monosaccharides or polysaccharides can be degraded. Such methods, in some embodiments, include the step of contacting a monosaccharide or polysaccharide or sample containing monosaccharides or polysaccharides with a 6-sulfatase or N-sulfamidase (or composition thereof) of the invention. The methods can further include steps whereby the sample or portion thereof is then analyzed with an analytic technique to determine the result of the contacting (e.g., the products formed or not formed).

In any of the methods provided the 6-O-sulfatase or N-sulfamidase can be used alone or with other polysaccharide-degrading enzymes, such as GAG-degrading enzymes. “GAG-degrading enzymes” refer to enzymes that degrade a glycosaminoglycan and include but are not limited to heparinase I, heparinase II, heparinase III, 2-O sulfatase, Δ4,5 glycuronidase, other sulfatases, glycosyl hydrolases, endoglucuronidases, α-glucosidase, β-glucosidase, other lyases, modified versions of these enzymes, variants and functionally active fragments thereof. In particular, in some embodiments, 6-O-sulfatase or N-sulfamidase can be used subsequent to or concomitantly with a heparinase, 2-O-sulfatase, Δ4,5 glycuronidase, or all or some combination of these enzymes, to degrade and/or analyze a monosaccharide or polysaccharide. In addition, in some embodiments, N-sulfamidase is used subsequent to or concomitantly with 6-O-sulfatase.

As used herein the terms “glycosaminoglycan” and “GAG” are used interchangeably to refer to a family of molecules, which include HSGAGs that are molecules having heparin-like/heparan sulfate-like structures and properties. These molecules include but are not limited to low molecular weight heparin (LMWH), heparin, biotechnologically prepared heparin, chemically modified heparin, synthetic heparin and heparan sulfate. The term “biotechnological heparin” encompasses heparin that is prepared from natural sources of polysaccharides which have been chemically modified and is described for example in Razi et al., Bioche. J. 1995 Jul. 15; 309 (Pt 2): 465-72. Chemically modified heparin is described in Yates et al., Carbohydrate Res (1996) Nov. 20; 294:15-27, and is known to those of skill in the art. Synthetic heparin is well known to those of skill in the art and is described in Petitou, M. et al., Bioorg Med Chem. Lett. (1999) Apr. 19; 9(8):1161-6.

In some embodiments, the GAG, such as a HSGAG, or monosaccharide thereof is or contains a N-sulfated or N-acetylated 6-O-sulfated glucosamine. In further embodiments, the GAG or monosaccharide thereof is sulfated or acetylated at the 2-amino position. In other embodiments the GAG or monosaccharide thereof does not contain 3-O sulfation, 6-O sulfation or does not contain both. In another embodiment, the GAG, such as a HSGAG, or monosaccharide thereof does not contain an unsubstituted amine on the same pyranose ring. In still another embodiment, the GAG, such as a HSGAG, does not contain a glycosidically linked uronic acid at the non-reducing C4 position. In yet another embodiment, the GAG, such as a HSGAG, or monosaccharide thereof is not unsaturated. In other embodiments, the GAG, such as a HSGAG, or monosaccharide thereof is any of the specific substrates used in the analysis described further below in the Examples.

The 6-O-sulfatase and N-sulfamidase of the invention may be used, for example, as a tool to sequence polysaccharides. Detailed methods for sequencing polysaccharides and other polymers are disclosed in U.S. patent application Ser. Nos. 09/557,997 and 09/558,137. These methods utilize tools such as GAG-degrading enzymes in the sequencing process. The 6-O-sulfatase and N-sulfamidase of the invention are useful as such a tool. Briefly, the method is performed by enzymatic digestion, followed by mass spectrometry and capillary electrophoresis. In the example described in the Rhomberg reference, enzymatic reactions are performed by adding 1 microliter of enzyme solution to 5 microliter of substrate solution. The digestion is then carried out at room temperature (22° C.), and the reaction is stopped at various time points by removing 0.5 microliter of the reaction mixture and adding it to 4.5 microliter of a MALDI matrix solution, such as caffeic acid (approximately 12 mg/mL) and 70% acetonitrile/water. The reaction mixture is then subjected to MALDI mass spectrometry. The MALDI surface is prepared by the method of Xiang and Beavis (Xiang and Beavis (1994) Rapid. Commun. Mass. Spectrom. 8, 199-204). A two-fold lower access of basic peptide (Arg/Gly)₁₅ is premixed with matrix before being added to the oligosaccharide solution. A 1 microliter aliquot of sample/matrix mixture containing 1-3 picomoles of oligosaccharide is deposited on the surface. After crystallization occurs (typically within 60 seconds), excess liquid is rinsed off with water. MALDI mass spectrometry spectra is then acquired in the linear mode by using a PerSeptive Biosystems (Framingham, Mass.) Voyager Elite reflectron time-of-flight instrument fitted with a 337 nanometer nitrogen laser. Delayed extraction is used to increase resolution (22 kV, grid at 93%, guidewire at 0.15%, pulse delay 150 ns, low mass gate at 1,000, 128 shots averaged). Mass spectra are calibrated externally by using the signals for proteinated (Arg/Gly)₁₅ and its complex with the oligosaccharide. Capillary electrophoresis may then be performed on a Hewlett-Packard^(3D) CE unit by using uncoated fused silica capillaries (internal diameter 75 micrometers, outer diameter 363 micrometers, 1_(det) 72.1 cm, and 1_(tot) 85 cm). Analytes are monitored by using UV detection at 233 nm and an extended light path cell (Hewlett-Packard). The electrolyte is a solution of 10 microliter dextran sulfate and 50 millimolar Tris/phosphoric acid (pH 2.5). Dextran sulfate is used to suppress nonspecific interactions of the glycosaminoglycan oligosaccharides with a silica wall. Separations are carried out at 30 kV with the anode at the detector side (reversed polarity). A mixture of a 1/5-naphtalenedisulfonic acid and 2-naphtalenesulfonic acid (10 micromolar each) is used as an internal standard.

Additionally, the coupling of CE and MALDI-MS with enzymes and a bioinformatics-based, property-encoded nomenclature (PEN) have led to a sequencing strategy (PEN-MALDI) described in (Venkataraman, G., Shriver, Z., Raman, R., and Sasisekharan, R. (1999) Science 286, 537-42). Other analytic techniques for use in sequencing (or analyzing generally) polysaccharides or monosaccharides are known in the art and include the use of 1 and 2D NMR, ion exchange HPLC, gel electrophoresis, etc. An analysing step of any of the methods provided herein can include the use of any of the analytic techniques listed herein or otherwise known in the art.

The 6-O-sulfatase and N-sulfamidase of the invention can also be used in other methods of analyzing monosaccharides, polysaccharides or samples containing them. Such methods include methods for identifying the presence of a particular monosaccharide or polysaccharide in a sample, methods for determining the purity of a monosaccharide or polysaccharide in a sample, methods for determining the composition of a polysaccharide in a sample, etc. A “sample”, as used herein, refers to any sample that may contain a monosaccharide or polysaccharide.

In another aspect of the invention, it was found that the activity of 6-O-sulfatase and N-sulfamidase can be affected by pH as well as the presence of sodium chloride, sulfate, phosphate, calcium or chelators. Therefore, compositions of 6-O-sulfatase and N-sulfamidase are provided in which the composition is of a certain pH. The pH can be for example greater than 4.5 or less than 9 or both. In some embodiments the pH is in the range of 5-9, 6-8 or 5.5-6.5. In still other embodiments, the pH is 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 or 9.

Compositions are also provided wherein the composition further comprises calcium. Compositions are further provided where in the composition does not comprise a chelator. In some embodiments, the compositions that do not contain a chelator also do not contain calcium. Chelators are known in the art and include, for example, EDTA and EGTA. In some embodiments, the chelator is at a concentration of less than 5 mM, less than 4 mM, less than 3 mM, less than 2 mM, less than 1.5 mM, less than 1 mM, less than 0.5 mM, etc. In some embodiments, the chelator is present at a concentration of 5 mM, 4 mM, 3 mM, 2 mM, 1.5 mM, 1 mM, or 0.5 mM, etc.

Compositions are further provided wherein the composition does not comprise sulfate ions, phosphate ions or both. In some embodiments, the sulfate ions are present at a concentrations of less than 20 mM, less than 19 mM, less than 18 mM, less than 17 mM, less than 16 mM, less than 15 mM, less than 14 mM, less than 13 mM, less than 12 mM, less than 11 mM, less than 10 mM, less than 9 mM, less than 8 mM, less than 7 mM, less than 6 mM, less than 5 mM, less than 4 mM, less than 3 mM, less than 2 mM, or less than 1 mM, etc. In other embodiments, the sulfate ions are present at a concentration of between 5-20 mM. In still other embodiments, the composition comprises phosphate ions at a concentration that is less than or equal to 5 mM, less than 4 mM, less than 3 mM, less than 2 mM, or less than 1 mM, etc.

Compositions are also provided wherein the composition has a concentration of sodium chloride that is less than 1 M, less than 0.9 M, less than 0.8 M, less than 0.7 M, less than 0.6 M, less than 0.5 M, less than 0.4 M, less than 0.3 M, less than 0.2 M, or less than 0.1 M, etc. In still other embodiments the concentration of sodium chloride is between 0.5 M and 1 M.

Compositions are also provided in which the composition further comprises acetate buffer.

In addition, methods of degrading or analyzing monosaccharides or polysaccharides with such compositions or in the presence or absence of the above compounds are also provided.

One of ordinary skill in the art, in light of the present disclosure, is enabled to produce substantially pure preparations of degraded monosaccharide or polysaccharide compositions utilizing the a 6-O-sulfatase or N-sulfamidase enzymes alone or in conjunction with other polysaccharide-degrading enzymes. The preparations can be prepared from HSGAG sources. A “HSGAG source” as used herein refers to heparin-like/heparan sulfate-like glycosaminoglycan compositions which can be manipulated to produce degraded products using standard technology, including enzymatic degradation, etc. As described above, HSGAGs include but are not limited to isolated heparin, chemically modified heparin, biotechnology prepared heparin, synthetic heparin, heparan sulfate, and LMWH. The HSGAGs sources can be natural sources, prepared by direct synthesis, etc.

The enzymatic compositions of the invention may also be used to remove active monosaccharides or polysaccharides (e.g., GAGs, such as HSGAGs) from a fluid (e.g., a GAG containing fluid, such as a HSGAG containing fluid). A fluid is contacted with the 6-O-sulfatase or N-sulfamidase of the invention, alone or in combination with other enzymes. The method is particularly useful for the ex vivo removal of GAGs, such as HSGAGs, from blood. In one embodiment of the invention the 6-O-sulfatase or N-sulfamidase is immobilized on a solid support as is conventional in the art. The solid support containing the immobilized 6-O-sulfatase or N-sulfamidase may be used in extracorporeal medical devices (e.g. hemodialyzer, pump-oxygenator) for systemic heparinization to prevent the blood in the device from clotting. The support membrane containing immobilized 6-O-sulfatase or N-sulfamidase is positioned at the end of the device to neutralize the GAG, such as a HSGAG, before the blood is returned to the body.

The 6-O-sulfatase or N-sulfamidase may be immobilized to any type of support but if the support is to be used in vivo or ex vivo it is desired that the support is sterile and biocompatible. A biocompatible support is one which would not cause an immune or other type of damaging reaction when used in a subject. The 6-O-sulfatase or N-sulfamidase may be immobilized by any method known in the art. Many methods are known for immobilizing proteins to supports. A “solid support” as used herein refers to any solid material to which a polypeptide can be immobilized.

Solid supports, for example, include but are not limited to membranes, e.g., natural and modified celluloses such as nitrocellulose or nylon, Sepharose, Agarose, glass, polystyrene, polypropylene, polyethylene, dextran, amylases, polyacrylamides, polyvinylidene difluoride, other agaroses, and magnetite, including magnetic beads. The carrier can be totally insoluble or partially soluble and may have any possible structural configuration. Thus, the support may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube or microplate well, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, bottom surface of a microplate well, etc.

It has been recognized that cells synthesize distinct GAG sequences and decorate themselves with these sequences, using the extraordinary information content present in the sequences to bind specifically to many signaling molecules and thereby regulate various biological processes. The compositions of the invention, therefore, have many therapeutic utilities. The invention is useful in a variety of in vitro, in vivo and ex vivo methods in which therapies are useful. The compositions include the enzymatic compositions provided as well as the compositions that contain a degraded GAG, such as a degraded HSGAG, or monosaccharide thereof. A “degraded GAG” as used herein refers to a molecule or molecules which are degraded GAGs or pieces or fragments thereof that have been degraded through the use of the enzymatic compositions provided herein. Such compounds may be generated using an enzymatic composition or they may be synthesized de novo. Degraded GAG fragments, such as degraded HSGAG fragments, including monosaccharides, can be tested for therapeutic activity using any of the assays described herein or known in the art.

The compositions of the invention can be used for the treatment of any type of condition in which such therapy has been identified as or can be determined to be a useful therapy, such as for treating coagulation disorders, treating cancer, treating inflammatory disorders, inhibiting angiogenesis, preventing neovascularization, inhibiting metastasis, regulating apoptosis, etc. One of ordinary skill in the art is enabled to prepare or identify an appropriate therapeutic composition, depending on the subject and the disorder being treated. These compositions may be used alone or in combination with other therapeutics.

The invention is useful for treating and/or preventing any disease/condition in a subject whereby glycosaminoglycans have been found to be important in the development and/or progress of the disease. The terms “treat” and “treating” as used herein refers to reversing or blocking the progression of the disease in the subject. Treating a disease also includes exacting a desired improvement in the disease or symptoms of the disease. For example to treat a subject with tumor cell proliferation refers to inhibiting completely or partially the proliferation or metastasis of a cancer or tumor cell, as well as inhibiting or preventing any increase in the proliferation or metastasis of a cancer or tumor cell.

A “subject having a disease” is a subject that can be diagnosed as having the disease, e.g., a person having cancer is identified by the presence of cancerous cells. A “subject at risk of having a disease” as used herein is a subject who has a high probability of developing the disease. These subjects include, for instance, subjects having a genetic abnormality, the presence of which has been demonstrated to have a correlative relation to a higher likelihood of developing the disease. For diseases brought about by exposure to disease causing agents, subjects at risk are those who are exposed to the disease causing agents such as tobacco, asbestos, chemical toxins, viruses, parasites, etc. A subject at risk also includes those who have previously been treated for the disease and have the possibility of having a recurrence of the disease. When a subject at risk of developing a disease is treated with a composition of the invention, the subject is able to prevent the occurrence of the disease or reduce the possibility of developing the disease.

The compositions are useful for treating or preventing disorders associated with coagulation. A “disease associated with coagulation” or “coagulation disorder” as used herein refers to a condition characterized by an interruption in the blood supply to a tissue due to a blockage of the blood vessel responsible for supplying blood to the tissue such as is seen for myocardial or cerebral infarction. A cerebral ischemic attack or cerebral ischemia is a form of ischemic condition in which the blood supply to the brain is blocked. This interruption in the blood supply to the brain may result from a variety of causes, including an intrinsic blockage or occlusion of the blood vessel itself, a remotely originated source of occlusion, decreased perfusion pressure or increased blood viscosity resulting in inadequate cerebral blood flow, or a ruptured blood vessel in the subarachnoid space or intracerebral tissue.

A disease associated with coagulation as used herein also is intended to encompass atherosclerosis. Atherosclerosisis a disease of the arteries whereby blood flow can be reduced due to the development of atheromatous plaques along the interior walls of the arteries. These plaques begin by the initial deposition of cholesterol crystals which grow larger with time. In addition to the cholesterol deposition, plaques also grow due to the proliferation of the surrounding cells. In time, the artery may become completely occluded due to this plaque growth.

The compositions may be used alone or in combination with a therapeutic agent for treating a disease associated with coagulation. Examples of therapeutics useful in the treatment of diseases associated with coagulation include anticoagulation agents, antiplatelet agents and thrombolytic agents.

Anticoagulation agents prevent the coagulation of blood components and thus prevent clot formation. Anticoagulants include, but are not limited to, heparin, warfarin, coumadin, dicumarol, phenprocoumon, acenocoumarol, ethyl biscoumacetate, and indandione derivatives.

Antiplatelet agents inhibit platelet aggregation and are often used to prevent thromboembolic stroke in patients who have experienced a transient ischemic attack or stroke. Antiplatelet agents include, but are not limited to, aspirin, thienopyridine derivatives such as ticlopodine and clopidogrel, dipyridamole and sulfinpyrazone, as well as RGD mimetics and also antithrombin agents such as, but not limited to, hirudin.

Thrombolytic agents lyse clots which cause the thromboembolic stroke. Thrombolytic agents have been used in the treatment of acute venous thromboembolism and pulmonary emboli and are well known in the art (e.g. see Hennekens et al, J Am Coll Cardiol; v. 25 (7 supp), p. 18S-22S (1995); Holmes, et al, J Am Coll Cardiol; v. 25 (7 suppl), p. 10S-17S (1995)). Thrombolytic agents include, but are not limited to, plasminogen, a₂-antiplasmin, streptokinase, antistreplase, tissue plasminogen activator (tPA), and urokinase. “tPA” as used herein includes native tPA and recombinant tPA, as well as modified forms of tPA that retain the enzymatic or fibrinolytic activities of native tPA. The enzymatic activity of tPA can be measured by assessing the ability of the molecule to convert plasminogen to plasmin. The fibrinolytic activity of tPA may be determined by any in vitro clot lysis activity known in the art, such as the purified clot lysis assay described by Carlson, et. al., Anal. Biochem. 168, 428-435 (1988) and its modified form described by Bennett, W. F. et al., 1991, J. Biol. Chem. 266(8):5191-5201, the entire contents of which are hereby incorporated by reference.

The compositions of the invention are also useful for inhibiting angiogenesis. An effective amount for inhibiting angiogenesis of the preparation is administered to a subject in need of treatment thereof. Angiogenesis as used herein is the inappropriate formation of new blood vessels. “Angiogenesis” often occurs in tumors when endothelial cells secrete a group of growth factors that are mitogenic for endothelium causing the elongation and proliferation of endothelial cells which results in a generation of new blood vessels. Several of the angiogenic mitogens are heparin binding peptides which are related to endothelial cell growth factors. The inhibition of angiogenesis can cause tumor regression in animal models, suggesting a use as a therapeutic anticancer agent. An effective amount for inhibiting angiogenesis is an amount of a preparation which is sufficient to diminish the number of blood vessels growing into a tumor. This amount can be assessed in an animal model of tumors and angiogenesis, many of which are known in the art.

The compositions of the invention are also useful for treating and preventing cancer cell metastasis. The invasion and metastasis of cancer is a complex process which involves changes in cell adhesion properties which allow a transformed cell to invade and migrate through the extracellular matrix (ECM) and acquire anchorage-independent growth properties (Liotta, L. A., et al., Cell 64:327-336, 1991). Some of these changes occur at focal adhesions, which are cell/ECM contact points containing membrane-associated, cytoskeletal, and intracellular signaling molecules. Metastatic disease occurs when the disseminated foci of tumor cells seed a tissue which supports their growth and propagation, and this secondary spread of tumor cells is responsible for the morbidity and mortality associated with the majority of cancers. Thus the term “metastasis” as used herein refers to the invasion and migration of tumor cells away from the primary tumor site.

The barrier for the tumor cells may be an artificial barrier in vitro or a natural barrier in vivo. In vitro barriers include but are not limited to extracellular matrix coated membranes, such as Matrigel. Thus, the enzymatic compositions or degradation products thereof can be tested for their ability to inhibit tumor cell invasion in a Matrigel invasion assay system as described in detail by Parish, C. R., et al., “A Basement-Membrane Permeability Assay which Correlates with the Metastatic Potential of Tumour Cells,” Int. J. Cancer, 1992, 52:378-383. Matrigel is a reconstituted basement membrane containing type IV collagen, laminin, heparan sulfate proteoglycans such as perlecan, which bind to and localize bFGF, vitronectin as well as transforming growth factor-β (TGF-β) urokinase-type plasminogen activator (uPA), tissue plasminogen activator (tPA), and the serpin known as plasminogen activator inhibitor type 1 (PAI-1). Other in vitro and in vivo assays for metastasis have been described in the prior art, see, e.g., U.S. Pat. No. 5,935,850, issued on Aug. 10, 1999, which is incorporated by reference. An in vivo barrier refers to a cellular barrier present in the body of a subject.

According to another aspect of the invention, there is provided methods for treating subjects having or at risk of having cancer. The cancer may be a malignant or non-malignant cancer. Cancers or tumors include but are not limited to biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas.

When administered to a patient undergoing cancer treatment, the compounds may be administered in cocktails containing other anti-cancer agents. The compounds may also be administered in cocktails containing agents that treat the side-effects of radiation therapy, such as anti-emetics, radiation protectants, etc.

Anti-cancer drugs that can be co-administered with the compounds of the invention include, but are not limited to, Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Fluorocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; and Zorubicin Hydrochloride.

The compositions of the invention can also be used to treat a subject with an inflammatory disorder. In some embodiments the inflammatory disorder is non-autoimmune inflammatory bowel disease, post-surgical adhesions, coronary artery disease, hepatic fibrosis, acute respiratory distress syndrome, acute inflammatory pancreatitis, endoscopic retrograde cholangiopancreatography-induced pancreatitis, burns, atherogenesis of coronary, cerebral and peripheral arteries, appendicitis, cholecystitis, diverticulitis, visceral fibrotic disorders, wound healing, skin scarring disorders (keloids, hidradenitis suppurativa), granulomatous disorders (sarcoidosis, primary biliary cirrhosis), asthma, pyoderma gandrenosum, Sweet's syndrome, Behcet's disease, primary sclerosing cholangitis or an abscess. In still another embodiment the inflammatory condition is an autoimmune condition. The autoimmune condition in some embodiments is rheumatoid arthritis, rheumatic fever, ulcerative colitis, Crohn's disease, autoimmune inflammatory bowel disease, insulin-dependent diabetes mellitus, diabetes mellitus, juvenile diabetes, spontaneous autoimmune diabetes, gastritis, autoimmune atrophic gastritis, autoimmune hepatitis, thyroiditis, Hashimoto's thyroiditis, insulitis, oophoritis, orchitis, uveitis, phacogenic uveitis, multiple sclerosis, myasthenia gravis, primary myxoedema, thyrotoxicosis, pernicious anemia, autoimmune haemolytic anemia, Addison's disease, scleroderma, Goodpasture's syndrome, Guillain-Barre syndrome, Graves' disease, glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid, sympathetic opthalmia, idiopathic thrombocylopenic purpura, idiopathic feucopenia, Siogren's syndrome, Wegener's granulomatosis, poly/dermatomyositis or systemic lupus erythematosus.

The compositions provided thus can also include anti-inflammatory agents. Anti-inflammatory agents include Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam; Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride; Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Salycilates; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin; Glucocorticoids; and Zomepirac Sodium.

HSGAGs (along with collagen) are key components of the cell surface-extracellular matrix (ECM) interface. While collagen-like proteins provide the necessary extracellular scaffold for cells to attach and form tissues, the complex polysaccharides fill the space created by the scaffold and act as a molecular sponge by specifically binding and regulating the biological activities of numerous signaling molecules like growth factors, cytokines etc. Therefore, the compositions provided herein can also be used in methods of repairing tissues.

Each of these disorders mentioned herein is well-known in the art and is described, for instance, in Harrison's Principles of Internal Medicine (McGraw Hill, Inc., New York), which is incorporated by reference.

The invention also encompasses screening assays for identifying other compounds for the treatment of a tumor and for preventing metastasis. The assays can be accomplished, for example, by treating a tumor or isolated tumor cells with an enzymatic composition as provided herein and isolating the resultant degraded GAGs, such as degraded HSGAGs. Thus, the invention encompasses individualized therapies, in which a tumor or portion of a tumor is isolated from a subject and used to prepare the therapeutic degraded GAGs, such as degraded HSGAGs. These therapeutic GAGs can be re-administered to the subject to protect the subject from further tumor cell proliferation or metastasis or from the initiation of metastasis if the tumor is not yet metastatic. Alternatively the GAGs can be used in a different subject having the same type or tumor or a different type of tumor.

The compounds provided herein may also be linked to a targeting molecule. A targeting molecule is any molecule or compound which is specific for a particular cell or tissue and which can be used to direct the compound to the cell or tissue. Preferably, in some embodiments, the targeting molecule is a molecule which specifically interacts with a cancer cell or a tumor. For instance, the targeting molecule may be a protein or other type of molecule that recognizes and specifically interacts with a tumor antigen.

Tumor-antigens include Melan-A/MART-1, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)—C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100^(Pmel117), PRAME, NY-ESO-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, cdc27, adenomatous polyposis coli protein (APC), fodrin, P1A, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, lmp-1, EBV-encoded nuclear antigen (EBNA)-1, and c-erbB-2.

Effective amounts of the compounds of the invention are administered to subjects in need of such treatment. Effective amounts are those amounts which will result in a desired improvement in the condition or symptoms of the condition, e.g., for cancer, this can be a reduction in cellular proliferation or metastasis, without causing other medically unacceptable side effects. Such amounts can be determined with no more than routine experimentation. It is believed that doses ranging from 1 nanogram/kilogram to 100 milligrams/kilogram, depending upon the mode of administration, will be effective. The absolute amount will depend upon a variety of factors (including whether the administration is in conjunction with other methods of treatment, the number of doses and individual patient parameters including age, physical condition, size and weight) and can be determined with routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. The mode of administration may be any medically acceptable mode including oral, subcutaneous, intravenous, etc.

In general, when administered for therapeutic purposes, the formulations of the invention are applied in pharmaceutically acceptable solutions. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

The compositions of the invention may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% W/V); citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V); and phosphoric acid and a salt (0.8-2% W/V). Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).

The present invention provides pharmaceutical compositions, for medical use, which comprise the compounds of the invention with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The term “pharmaceutically-acceptable carrier” as used herein, means one or more compatible solid or liquid filler, diluants or encapsulating substances which are suitable for administration to a human or other animal. In the present invention, the term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being comingled with the compounds of the present invention or other compositions, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular active agent selected, the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of an immune response without causing clinically unacceptable adverse effects. A preferred mode of administration is a parenteral route. The term “parenteral” includes subcutaneous injections, intravenous, intramuscular, intraperitoneal, intra sternal injection or infusion techniques. Other modes of administration include oral, mucosal, rectal, vaginal, sublingual, intranasal, intratracheal, inhalation, ocular, and transdermal, etc.

For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the polymer into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. The compositions may be stored lyophilized.

Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the heparinases of the invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as polylactic and polyglycolic acid, polyanhydrides and polycaprolactone; nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like. Specific examples include, but are not limited to: (a) erosional systems in which the polysaccharide is contained in a form within a matrix, found in U.S. Pat. No. 4,452,775 (Kent); U.S. Pat. No. 4,667,014 (Nestor et al.); and U.S. Pat. No. 4,748,034 and U.S. Pat. No. 5,239,660 (Leonard) and (b) diffusional systems in which an active component permeates at a controlled rate through a polymer, found in U.S. Pat. No. 3,832,253 (Higuchi et al.) and U.S. Pat. No. 3,854,480 (Zaffaroni). In addition, a pump-based hardware delivery system can be used, some of which are adapted for implantation.

A subject is any human or non-human vertebrate, e.g., dog, cat, horse, cow, pig.

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

EXAMPLES Materials and Methods

Reagents

Fluorescent glucopyranoside substrates 4-methyllumbelliferyl-α/β-D-glucopyranoside (4-MU-α-D-Glc, 4-MU-β-D-Glc) were purchased from EMD Biosciences, Inc (San Diego, Calif.). 6-O and N-sulfated fluorogenic glycopyranoside derivatives were obtained through Toronto Research Chemicals (Toronto, Canada). Glucosamine and galactosamine monosaccharides, arylsulfate substrates 4-catechol-sulfate and 4-methyllumbelliferyl-sulfate were purchased from Sigma Chemical Co. (St. Louis, Mo.). Exoglucosidases were purchased from MP Biomedicals (Irvine, Calif.). Materials for genomic library construction and screening were obtained from Stratagene (La Jolla, Calif.). PCR enzymes, TOP10 chemically competent cells, and oligonucleotide primers were obtained from Invitrogen (Carlsbad, Calif.). Additional molecular cloning reagents were purchased from New England Biolabs (Beverly, Mass.) or the manufacturers listed.

Molecular Cloning of Flavobacterial 6-O-Sulfatase and N-Sulfamidase

Both flavobacterial sulfatase genes were cloned by PCR from a λZAPII flavobacterial genomic library originally screened using DNA hybridization probes specific to 2-O-sulfatase. Library construction, hybridization screening, and phage excision were as described. Two overlapping clones were expanded by chromosomal walking and restriction mapping using the Lambda DASH II genomic cloning kit (Stratagene, La Jolla, Calif.) for the ligation of size fractionated genomic DNA (generated by a partial Sau3A I digestion). 2-O-sulfatase positive clones from an amplified library were plaque purified through three successive rounds and the DNA purified from a high titer lysate using standard techniques (Ausubel et al., eds. 1987 Current Protocols in Molecular Biology, John Wiley and Sons, NY, N.Y.). For DNA sequencing, recombinant phage DNA was subcloned into pBluescript SK+/− (Stratagene, La Jolla, Calif.). The coding sequences of two putative sulfatase genes (described as ORF B and ORF C) were identified by the canonical PFAM sulfatase family identifier (CXPXRXXXXS/TG; SEQ ID NO: 5) and subsequently PCR amplified using the following primer sets: 1) for ORF B (6-O-sulfatase), 5′ GAA TTC ATA TGG GTA AAT TGA AAT TAA TTT TA 3′ (forward; SEQ ID NO: 11) and 5′ GGA TCC TCG AGT TAT AAA GCT TCA GTT GGA TTC GT 3′ (reverse; SEQ ID NO: 12) for ORF C(N-sulfamidase), 5′ TCT AGA CAT ATG AAA TTT AAC AAA TTG AAA TAT TTC 3′ (forward; SEQ ID NO: 13) and 5′ GGA TCC TCG AGT TAC TTC AAA TAA TTG TAA CTG GAA T 3′ (reverse; SEQ ID NO: 14). Amplified genes were subcloned into the T7-based bacterial expression vector pET28a (Novagen, San Diego, Calif.) as an Nde 1-Xho 1 cassette (restriction sites underlined). Cloning, as such, allowed these genes to be expressed as an NH₂-terminal 6×His fusion with an intervening thrombin cleavage site for facile removal of this tag following protein purification.

Bacterial Expression and Protein Purification

Recombinant protein expression in the E. coli strain BL21(DE3) and one-step affinity purification by nickel chelation chromatography were as described for 2-O-sulfatase (Myette et al., 2003 J Biol Chem 278, 12157-12166). The prediction of NH₂-terminal signal sequences and putative cleavage sites for the two respective proteins was made by the Von Heijne computational method (Nielsen et al., 1997 Protein Eng 10, 1-6.). Engineering and expression of these truncated proteins (minus signal sequences) were as described above for the full-length genes with the exception of substituting the 5′ primers which were used in the original PCR amplification step. These internal primers included 5′ TCT AGA CAT ATG TCT TGC CAG CAG CCT AAA C 3′ (for ORF B; SEQ ID NO: 15) and 5′ TCT AGA CAT ATG TCC TGC ACT TCG CCG GAA 3′ (for ORF C; SEQ ID NO: 16), with the Nde 1 site underlined. As such, the ORF B gene sequence begins at Met 18 (FIG. 1). Likewise, the ORF C gene sequence begins at Ser 21 (FIG. 2). Removal of the 6×His tag was achieved by site-specific protease cleavage using the thrombin cleavage capture kit (Novagen, San Diego, Calif.). Proteolysis conditions were generally as described for other recombinantly expressed flavobacterial heparin degrading enzymes. Following concentrating the enzymes by ultrafiltration, cleaved proteins were dialyzed overnight against 4 liters of 50 mM Tris, pH 7.5 and 0.1 M NaCl, 4° C., using a 3 mL slide-a-lyzer cassette with a 10,000 molecular weight cutoff (MWCO) (Pierce Chemical, Rockland, Ill.).

Final protein concentrations were determined colorimetrically using the Bradford Assay (Bio-Rad, Hercules, Calif.) and confirmed by UV absorption spectroscopy using theoretical molar extinction coefficients (ε₂₈₀) of 94,730 M⁻¹ (61,572 Da) and 86,340 (53,193 Da) for the NH₂-terminally truncated ORF B (6-O-sulfatase) and ORF C(N-sulfamidase), respectively. These values were calculated for thrombin-cleaved proteins lacking a 6×His purification tag. Enzymes were stored at 4° C. at a concentration of ˜10 mg/mL. Full enzyme activity was retained for several months under these conditions.

Arylsulfatase Assay

Arylsulfatase activity was measured independently using two chromogenic substrates, 4-catechol-sulfate and 4-methylumbelliferyl-sulfate (4-MUS). The catechol substrate assay was conducted generally as described (Beil et al., 1995 Eur J Biochem 229, 385-394). Briefly, 10 mM substrate were incubated with ˜30 μM recombinant enzyme overnight (12-15 hours) at 37° C. in a 100 μL reaction which included 50 mM MES pH 7.0 and +2 mM CaCl₂. Reactions were quenched by the addition of 5 μL 5M NaOH and colorimetric activity determined spectroscopically at 515 nm. Fluorimetric arylsulfatase assay using 4-MUS was as described (Morimoto-Tomita et al., 2002 J Biol Chem 277, 49175-85) with some modifications. Reaction conditions included 10 μM enzyme, 2 mM 4-MUS, 50 mM sodium acetate, pH 6.0, and 5 mM CaCl₂ in a 20 μL reaction volume. The enzyme incubation temperature was 30° C. Activity was measured as a function of time ranging from 3 to 24 hours; the reactions were quenched by the addition of 200 μL 0.5 M Na₂CO₃, pH 10.7. Detection of fluorescent methylumbelliferone was measured at this alkaline pH using a SpectraMax microtiter plate reader (Molecular Dynamics, Sunnyvale, Calif.) set at excitation and emission wavelengths of 360 and 440 nm, respectively. Fluorescence intensity was corrected against background (minus enzyme control). In both assays, 0.5 unit of arylsulfatase from Aerobacter aerogenes (Sigma, St. Louis, Mo.) was used as a positive control.

Pilot 6-O-Sulfatase and N-Sulfamidase Assays

Initial assessment of substrate specificity and pH optima was made using a capillary electrophoresis-based assay for the detection of desulfated products. Preliminary enzyme activity was measured against the following series of fluorescently derivatized, monosulfated gluco and galactopyranosides: 4-MU-GlcNAc,6S, 4-MU-GlcNS, 4-MU-GalNAc,6S and 4-MU-Gal6S. Standard reactions included 1 mM substrate, 1-10 μM enzyme, 50 mM sodium acetate, pH 5.5-6.5 and 5 mM CaCl₂ in a 20 μL reaction volume. For pilot experiments, exhaustive reactions involved overnight incubations at 30° C. Enzyme was inactivated by heat denaturation at 95° C. for 10 minutes, followed by a 10-fold dilution into water. Reaction products were resolved by capillary electrophoresis using a 25 cm long, 75 μm (i.d.) fused silica capillary (Agilent Technologies, Palo Alto, Calif.). Electrophoresis was carried out under negative polarity by applying a voltage of −15 kV (˜1.2 W) for 10 minutes. Substrate desulfation was measured as a percentage of substrate depletion relative to a minus enzyme control as monitored by the loss of UV absorbance at 315 nm detected at approximately 4 minutes. A standard capillary electrophoresis buffer included 50 mM Tris and 10 μM dextran sulfate (average MW of 10,000 Da) adjusted to pH 2.0 with phosphoric acid. At this pH, saccharide migration occurred on the basis of charge without the influence of electro-osmotic flow.

The effect of pH was likewise measured by capillary electrophoresis using the following three sets of buffers with overlapping pH ranging from 4.5 to 8.0:50 mM sodium citrate at 4.5, 5.0, and 5.5; 50 mM MES at 5.5, 6.0, 6.5 and 7.0; and 50 mM MOPS at 6.5, 7.0, 7.5, and 8.0. Reactions included 1 μM enzyme, 2 mM 4-MU-GlcNAc,6S (for 6-O-sulfatase) or 4-MU-GlcNS (for N-sulfamidase), 50 mM buffer and 5 mM CaCl₂ in a 20 μL reaction volume. Assay was initiated by the addition of 2 μl of a 10× enzyme stock to 18 μL of preheated reaction mixture. Reactions were carried out at 30° C. for either 30 minutes (N-sulfamidase assay) or 60 minutes (6-O-sulfatase assay) and quenched by heat and dilution as described above.

The ability of both enzymes to desulfate unsaturated heparin and chondroitin disaccharides was assessed essentially as described for capillary electrophoresis based compositional analyses of enzymatically generated glycosaminoglycan di- and tetra-saccharides (Venkataraman et al., 1999 Science 286, 537-542). For these studies, the following disaccharide substrates were tested: ΔUGlcNAc,6S; ΔU2SGlcNAc,6S; ΔUGlcNS; ΔUGlcNS,6S; ΔU2SGlcNS,6S; and ΔUGalNac,6S. Reactions included 500 μM substrate, 10 μM enzyme, 50 mM sodium acetate, pH 6.5 and +/−2 mM CaCl₂ in a 20 μL reaction volume.

Coupled Enzyme Assay for the Determination of Biochemical Reaction Conditions and Steady-State Kinetics

Indirect measurement of enzyme activity was also made using a fluorimetrically-based plate assay in which the prerequisite desulfation of the appropriate glucopyranoside 1→4 methlybelliferone substrate by either the 6-O-sulfatase or N-sulfamidase was coupled to the glucosidase-mediated hydrolysis of the stereo-specific 1→4 glycosidic linkage between the pyranose ring and the adjoining fluorophore. Release of the free fluorophore (4-MU) was monitored spectroscopically as described above for the arylsulfatase assay using 4-MU sulfate. The two sulfatase assays differed in the choice of substrate in accordance to their specificity as well as the glucosidase added during the second, rate-limiting step. For the 6-O-sulfatase, the hydrolysis of the substrate 4-MU-β-D-GlcNAc,6S at the 6-OH position was coupled to β-glucosidase purified from sweet almonds (MP Biomedicals, Solon, Ohio, Catalog No. 195197). Likewise, N-sulfamidase hydrolysis of 4-MU-α-D-GlcNS at the 2-amino position was coupled to α-glucosidase (MP Biomedicals, Catalog No. 153487). In both cases, the efficacy of the coupled assay was contingent on the intrinsically poor ability either glucosidase possesses for hydrolyzing the glycosidic bond when the adjoining glucosamine is modified by a sulfate. The presumption of the first (sulfatase) activity being the rate-limiting step was established experimentally. Reaction conditions were optimized to satisfy three related criteria: 1) linear readout of fluorescent signal which is directly proportional to sulfatase activity; 2) quantitative release of 4-MU by glucosidase activity under every biochemical condition examined and; 3) negligible fluorescent quenching of the free chromophore.

For the 6-O-sulfatase/β-glucosidase assay, the standard reaction conditions included 2 μM recombinant enzyme, 50 mM sodium acetate buffer, pH 5.5, and 5 mM CaCl₂ in a 20 μl reaction volume. 4-MU-GlcNAc,6S substrate concentration was varied from 0.1-2 mM. 2 μL of enzyme were added to each well of a microtiter plate (prechilled on ice), plate gently vortexed, and contents of each well spun down for 1 minute at 500×g, 4° C. The assay was initiated by transferring the 96 well plate to a heating block prequilibrated at 30° C. Sulfatase incubation (first enzyme) was carried out at 30° C. for 20 minutes, after which enzyme activity was inactivated by heat denaturation (95° C., 10 minutes). In the second enzyme (glucosidase) step, the microtiter plate was once again chilled on ice. 40 units of β-glucosidase was added to each well, the plate mixed by gentle vortexing, spun down at 500×g for 1 minute at 4° C. and transferred to a heating block prequilibrated at 37° C. Incubation proceeded for 60 minutes prior to being quenched with 200 μL 0.5 M Na₂CO₃, pH 10.7. Reactions were transferred to a black 96 well, flat-bottom FIA-plate and fluorescence measured as described above for the detection of free 4-MU. Fluorescent signal was adjusted to background (minus sulfatase control). For β-glucosidase, this background hydrolysis was somewhat dependent on initial 4-MU-GlcNAc,6S concentration, but typically was less than 10%. Molar conversion of product was extrapolated from a standard curve generated from varying concentrations of 4-MU from 0-300 μM.

Coupled N-sulfamidase assay was generally described for the 6-O-sulfatase, but with the following modifications: 4-MU-GlcNS as substrate, 50 mM sodium acetate at pH 6.0 (instead of 5.5) and 1 μM enzyme. For the second enzyme step, 5 units of α-glucosidase were added. Enzyme incubation was carried out for 22 hours at 37° C. The obvious difference in enzyme efficacies between α-glucosidase versus β-glucosidase is reflected in the substantially longer incubation times required for the α-glucosidase to quantitatively hydrolyze the glycosidic α1→4 linkage between the fluorophore and the desulfated glucosamine. All other reaction conditions were as described for the coupled 6-O-sulfatase/β-glucosidase assay.

Michaelis-Menten kinetics were extrapolated from V_(o) vs. substrate concentration plots fit by non-linear regression to pseudo-first order kinetics. Data represent the average of three experiments. Substrate concentration was varied from 0.1 to 2 mM 4-MU-GlcNAc,6S (for 6-O-sulfatase kinetics) or 4-MU-GlcNS (for N-sulfamidase kinetics). Additional conditions included the presence of 0, 0.5 mM, or 5 mM CaCl₂ or 1 mM EDTA under otherwise standard conditions described.

Compositional Analyses of Sulfatase Treated Heparin

20 μg heparin were preincubated with 10 μM 6-O-sulfatase or N-sulfamidase for 8 hours at 30° C. in a 20 μL reaction which included 25 mM sodium acetate, pH 7.0 and 2 mM calcium acetate, pH 7.0. Following this preincubation, enzymes were inactivated by heat denaturation at 95° C. for 10 minutes and heparin exhaustively digested overnight at 37° C. by the addition of 2 μL of a concentrated enzyme cocktail containing heparinase I and III. Subsequent CE-based compositional analyses of heparinase-derived disaccharides were completed as described (Venkataraman et al., 1999 Science 286, 537-542).

Sequential Degradation of Heparin Oligosaccharide by Flavobacterial Exo-Enzymes

The purified pentasulfated tetrasaccharide ΔU_(2S)H_(NS,6S)IH_(NS,6S) was provided by Dr. I. Capila (Momenta Pharmaceuticals, Cambridge, Mass.). Enzyme sequence was as follows: 2-O-sulfatase→Δ4,5 glycuronidase→6-O-sulfatase→N-sulfamidase. After each step, the enzyme was heat inactivated and 20 μL aliquots removed prior to the addition of the next enzyme in the sequence. Initial reaction conditions included 20 mM Tris, pH 7.2 and 60 nanomoles of tetrasaccharide in a 120 μL reaction volume. All enzyme reactions were carried out at 30° C. Enzyme specific conditions included the following: 1) 2-O-sulfatase, 1 μM enzyme, 6 hours; 2) Δ4,5 glycuronidase, 1 μM enzyme, 6 hours; 3) 6-O-sulfatase, 5 μM enzyme, 5 mM CaCl₂, 12-15 hours; 4) N-sulfamidase, same conditions as for 6-O-sulfatase. Molecular masses of enzyme products were determined by MALDI mass spectrometry using established methods (Rhomberg et al., 1998, Proc Natl Acad Sci USA 95, 12232-12237).

CE-LIF for the Detection of Sequentially Degraded Oligosaccharides

The APTS derivitization protocol was adapted from Chen et al. (Chen F. T. A., and Evangelista R. A. (1995) Analytical Biochemistry 230, 273-280). Briefly, 2 μl of 100 mM 9-aminopyrene-1,4,6-trisulfonate (APTS) in 25% acetic acid (v/v) were mixed with 10 μl of 1M sodium cyanoborohydride in tetrohydrofuran and 1 μmol of saccharide. The reaction mixture was incubated at 75° C. for 2 hr and was diluted 1:100 prior to CE analysis. CE-LIF was performed on a Beckman Coulter ProteomeLab PA 800 with 488 nm argon LIF module. Samples were loaded onto a N—CHO capillary (50 μm I.D.×65 cm total length) using 0.5 psi of pressure at the anode for 20 sec. Electrophoretic separations were performed using a 20 kV potential in a 100 mM sodium borate, pH 10.2 buffer for 15 min at 25° C. Fluorescence emission spectra were collected using a 520 nm narrow band filter.

ESI-Mass Spectrometry of Sulfated Glucosamine Monosaccharides

Electrospray ionization mass spectrometry was performed in the negative ion mode using an Agilent 1100 Series VL LC/MSD Trap. For simplicity, the samples were prepared by adding MeOH directly to the enzymatic reaction mixtures without purification and were directly injected into the source of the mass spectrometer using a syringe pump at a rate of ˜8 μl/min, The SPS function of the software (LC/MSD Trap Software 4.1 Build 143, MSD Trap Control Version 5.0 Build 65) was used to tune the instrument with the target mass set to the mass of the substrate, the sample stability set to 50% and the drive level set to 100%. Data were acquired over the scan range of 100-2200 m/z by accumulating 30,000 ions per scan. Capillary voltage was set to 3000 V. Nitrogen was used as the drying gas while helium was used as the nebulizing gas, with flow rates of 5 and 15 liters/min, respectively. In each case a minimum of ten spectra were averaged.

For the initial substrate specificity experiments on unlabeled monosaccharides, reactions were carried out with 2.5 mM substrate, 2.5 mM CaCl₂, excess enzyme and 37.5 mM Tris Buffer at pH 7.5 at 37° C. overnight. For the experiments determining the order of action of the enzymes, reactions were carried out with 100 μM substrate, 2 mM CaCl₂, 5 μM enzyme and various buffers at 37° C. overnight. Samples were diluted 1:10 in MeOH prior to analysis in the ESI, and the carrier solvent was H2O:MeOH (1:10, v/v). For the time course experiments showing desulfation of different unlabeled monosaccharides, reactions were carried out with 2.5 mM substrate, 5 mM CaCl₂, 1 μM enzyme and 50 mM acetate buffer at pH 5.5 for the 6-O-sulfatase and pH 6.0 for the N-sulfamidase at 37° C. Reactions were quenched by diluting the samples 1:4 in MeOH. The carrier solvent was H2O:MeOH (1:4, v/v). In these experiments, 4-MU-GlcNS was added prior to injection as an internal standard to monitor ionization efficiency and mass accuracy within the source and trap.

Results

Molecular Cloning and Recombinant Expression of Flavobacterium Heparinum Sulfatase Genes

The two sulfatase genes were identified through the screening of a genomic library with hybridization probes directed toward the flavobacterial 2-O-sulfatase. Two overlapping phagemid clones identified during this process were expanded by chromosomal walking and restriction mapping. Sequence analyses of this genomic region revealed two sizeable open reading frames of 1647 and 1524 base pairs (described hereafter as ORF B and ORF C, respectively). The two gene sequences putatively encode proteins of 545 (FIG. 1) and 500 (FIG. 2) amino acids in length (starting at the initiating Met). Neither sequence possessed an obvious Shine-Dalgarno ribosomal binding site within 10 nucleotides of the initiating ATG codon. A closer examination of their individual sequences at the protein level noted several important features. Both flavobacterial ORFs possess a N-terminal hydrophobic signal peptide and corresponding cleavage site sequence predicted by the Von Heijne method for gram-negative bacteria (Nielsen, H., et al. (1997) Protein Eng 10, 1-6). Both genes encode basic protein sequences of comparable amino acid composition (by mol percent). Of the two proteins, the ORF B gene product possesses a slightly higher theoretical pI (8.6 vs. 8.0) relative to ORF C. Both gene products also possess a canonical sulfatase domain as described by the Protein Family (PFAM) identifier PF 000884.

Both putative sulfatase genes were robustly expressed in E. coli as soluble enzymes. To achieve satisfactory expression levels, however, the removal of the amino terminal signal sequences of both proteins was genetically engineered. Exclusion of this domain, however, had little deleterious effect on each enzyme's specific activity. At the same time, replacement of this NH₂-terminal peptide with a histidine (6×His) tag facilitated purification of the recombinant proteins in essentially a single chromatographic step (to greater than 80% purity). Subsequent thrombin cleavage of the histidine tag was carried out. These ΔNH₂-terminal truncations (lacking both the native signal sequence and NH₂-6×His tag) were used in all subsequent biochemical characterizations of the two sulfatases. The apparent molecular weights of the two recombinant proteins based on SDS-PAGE were consistent with their theoretical molecular weights calculated from their respective amino acid compositions (ORF B, 61,572 Da; ORF C, 53,193 Da).

Biochemical Characterization of Recombinant HSGAG Sulfatases: Preliminary Determination of Monosaccharide Substrate Specificity

As a first step in the biochemical characterization of these enzymes, the possibility of both these enzymes (as well as the previously characterized 2-O-sulfatase) functioning as generic arylsulfatases was examined. All three enzyme activities were tested against 4-catechol sulfate and 4-MU-sulfate, two different aromatic sulfate esters commonly used as substrates to make this assessment. Of the three enzymes, only the 2-O-sulfatase exhibited an appreciable level of hydrolytic activity relative to a known arylsulfatase from Aerobacter aerogenes which served as a positive control. At the same time, the ORF B sulfatase did partly hydrolyze the 4-MU sulfate at a discernible rate, which was at least 3-fold greater than that measured for the ORF C protein, which exhibited only negligible activity.

The fact that the ORF B and ORF C encoded enzymes are both poor arylsulfatases does not preclude them from acting on sulfated carbohydrates. In reality, many such sulfatases (including those which desulfate heparin/heparan sulfate) fail to be classified as so-called “arylsulfatases” on the basis of this rather non-specific biochemical screen. Based at least in part on the multiple sequence alignment, it was thought that two additional glycosaminoglycan sulfatases, namely the heparan N-acetylglucosamine-6-O-sulfatase and heparan N-sulfamidase, had indeed been cloned. To further test these enzymes and examine their ability to act on carbohydrates, such as heparin and heparan sulfate, a modified substrate whereby the sulfated hexosamine was linked 1→4 (α or β) to methylbelliferone (4-MU) was used. The presence of this chromophore allowed for the direct monitoring of desulfation of the monosaccharide by capillary electrophoresis. Four monosulfated substrates were tested, all of which were commercially available. These included the two “heparin” monosaccharides 4-MU-GlcNAc,6S and 4-MU-GlcNS in addition to the 6-O-sulfated galactose sugars 4-MU-Gal6S and 4-MU-GalNAc,6S (corresponding to the monsaccharide constituents of keratan sulfate and chondroitin/dermatan sulfate, respectively). In this analysis, the ORF B sulfatase was specific for the 6-O-sulfated glucosamine (FIG. 5A). The ORF C gene product could only hydrolyze the glucosamine sulfated at the 2-amino position. The recombinantly expressed ORF B gene product did not act upon either of the two 6-O-sulfated galactose sugars. The structural discrimination for the two enzymes observed for the fluorescently derivatized sugars was confirmed by mass spectrometry using non-derivatized monosaccharides possessing otherwise identical chemistries.

The substrate specificities of the two flavobacteria-derived sulfatases was further investigated by examining the influence of various substitutions at the 2-amino, 3-OH and 6-OH positions of the glucosamine. In these experiments, desulfation of non-derivatized monosaccharide substrates was detected and quantified by electrospray ionization (ESI) mass spectrometry. The 6-O-sulfatase required a substituted amine (acetate or sulfate) at the 2-amino position. A comparative kinetic analysis of the two corresponding substrates (GlcNAc,6S vs. GlcNS,6S) indicated only a modest preference of the enzyme for the monosulfated substrate (FIG. 5B). Reciprocally related to this observation (FIG. 6), it was found that the N-sulfamidase activity at the 2-amino position was absolutely abolished when a second sulfate at the 6-O-sulfate was also present. Both hydrolases were completely inhibited by the presence of a 3-O-sulfate.

Optimization of In Vitro Reaction Conditions

Having identified chromogenic substrates for each recombinant sulfatase, these substrates were also used to further develop a fluorescence-based plate assay as the means to define the optimal in vitro reaction conditions for each sulfatase. Parameters, such as ionic strength, and the effect of divalent metal ions as well as steady-state enzyme kinetics were investigated. A coupled enzyme assay in which the recombinant sulfatase served as the primary (product limiting) enzyme and either α or β-glucosidase as the secondary enzyme was chosen. Use of this second enzyme permitted the indirect detection of relative sulfatase activity by means of the stoichiometric release of free 4-MU which served as the fluorescent signal. This coupled assay for both enzymes was validated in control experiments demonstrating only modest hydrolysis of the 1→4-MU glycosidic linkage of the sulfated glucosamine (6-O or NS) by either glucosidase.

Both the 6-O-sulfatase and the N-sulfamidase were sensitive to increasing ionic strength as measured by the addition of NaCl (FIG. 7A). For both enzymes, 50% inhibition was observed at approximately 200 mM NaCl with less than 20% activity remaining at 1 M NaCl relative to the zero NaCl control. Both enzymes exhibited slightly acidic pH optima (between 5.5 and 6.5) (FIGS. 7B and D). Of the two enzymes, the N-sulfamidase was active over a broader range, especially above pH 7.0. Neither enzyme was active below pH 4.5. The enzymes showed higher activity in acetate buffer when compared to sulfonate buffers such as MES and MOPS when examined over this same pH range. However, only the 6-O-sulfatase was inhibited by the addition of sulfate or phosphate (FIG. 7B). Of the two anions, phosphate was clearly a more effective inhibitor, with 50% inhibition of 6-O-sulfatase activity observed at approximately 2 mM PO₄ ²⁻ compared with approximately 20 mM SO₄.

Other members of this enzyme family share a relatively conserved active site and a common enzyme mechanism for sulfate hydrolysis (Parente et al., 1997 Curr Opin Genet Dev 7, 386-39; Bond et al., 1997 Structure 5, 277-289). Histidine, for example, is a candidate for participating in enzyme catalysis. At least two catalytic roles have been proposed for separate histidines based on crystallographic studies. The first role is stabilizing the Oγ2 oxygen of the hydrated formylglycine through hydrogen bonding, while also possibly acting as a proton acceptor. A second histidine stabilizes the sulfate itself, likewise through hydrogen bond contacts with one of the terminal oxygen atoms. Local sequence alignments between each of the flavobacterial sulfatases and the three structurally defined sulfatases (human arylsulfatase A (Lukatela et al., 1998) Biochemistry 37, 3654-3664; Waldow et al., 1999 J Biol Chem 274, 12284-12288) and B (Bond et al., 1997 Structure 5, 277-289) and the sulfatase from P. aeriginosa (Boltes et al., 2001 Structure (Camb) 9, 483-491) suggest histidine 130 for the 6-O-sulfatase as at least one of the homologous active site residues serving in this capacity.

In addition to histidine, other residues which line this consensus active site include at least three additional basic residues, which appear to form a binding pocket of positive ions. Two of these positively charged residues interact electrostatically with the negatively charged oxyanions of the sulfate; a third appears to interact with the hydrated formylglycine via a hydrogen bond. Potential homologous positions in the primary sequences of the two flavobacterially derived enzymes are, for example, depicted in FIG. 11 or as elsewhere described herein.

Role of Divalent Metal Ions in Enzyme Catalysis

Both sulfohydrolase activities were activated by the presence of calcium in a concentration-dependent manner albeit to clearly differing extents (FIG. 8). This divalent metal ion effect was especially pronounced for the N-sulfamidase which, in these experiments, required calcium for activity (FIG. 8B). In comparison, the 6-O-sulfatase, although activated 2-3 fold by the presence of calcium, was somewhat active even in the presence of 1 mM EDTA (FIG. 8A). Interestingly, the divalent metal activation for both enzymes was specific to calcium; inclusion of Mg⁺² or Mn⁺² had only negligible effects. To further examine this metal selectivity, the potential for enzyme inhibition in the presence of the calcium specific chelator, EGTA, was measured. EGTA was found to inhibit calcium-dependent N-sulfamidase activity (at 5 mM Ca²⁺) in a concentration-dependent manner, with 50% inhibition occurring at approximately 3 mM EGTA. In contrast, EGTA had no appreciable effect on 6-O-sulfatase specific activity when measured under the same conditions.

In an attempt to determine the mechanism by which calcium exerts its effect on the two HSGAG sulfatases, the effect of calcium on enzyme steady-state kinetics was measured (FIGS. 8C and 8D). Consistent with the previous results, the initial rates of both enzymes were affected by calcium in a concentration dependent fashion. For the 6-O-sulfatase, this was largely manifested as a k_(cat) effect (Table 1). For the N-sulfamidase, the effect of calcium was predictably more pronounced, with both kinetic parameters being affected in a generally proportional fashion. The catalytic role for calcium is supported by the consensus structure of the enzyme active site. In this snapshot, a divalent metal ion coordinates with at least one sulfate oxygen of the substrate while also coordinating with the carboxylates of four highly conserved acid residues surrounding the modified cysteine (FGly). Presumably, this coordination would promote catalysis by properly orienting the sulfate ester/amide bond for hydrolysis. Candidates are as predicted in FIG. 11 or as elsewhere described herein.

TABLE 1 Steady state kinetic parameters using 4-MU monosaccharide substrates 6-O-sulfatase N-sulfamidase Addition k_(cat) (min⁻¹) Km (μM) k_(cat)/Km(×10²) k_(cat) (min⁻¹) Km (nM) k_(cat)/Km (×10²⁾ none 2.5 146 1.7 N.D.* N.D.* N.D.* 0.5 mM Ca⁺² 3.3 217 1.5  5.1  45 11.3 5 mM Ca⁺² 6.8 327 2.1 21.9 178 12.3 1 mM EDTA 2.5 264 0.95 N.D.* N.D.* N.D.* *N.D. not determined due to lack of activity Oligosaccharides Substrates

The standard sulfatase assay described typically used singly sulfated, fluorogenic monosaccharides as substrates. The use of these enzymes to desulfate heparin/heparan sulfate oligosaccharides in accordance with their defined substrate specificities, however, was also explored. In nature, these HSGAG oligosaccharides could possess either an uronic acid (even number of saccharide units) or hexosamine (off number oligosaccharide) at their non-reducing ends. In the former case, the uronic acid would likely be unsaturated due to the preceding action of heparin lyases which cleave the GAG chain through a β-eliminative catalytic mechanism. To address this, both enzymes were initially tested against a panel of unsaturated heparin disaccharides. These included ΔU_(±2S)H_(NAc,6S) and ΔU_(±2S)H_(NS,6S) for the 6-O-sulfatase and ΔU_(±2S)H_(NS+6S) for the N-sulfamidase. For these experiments, standard reaction conditions were chosen as defined in the monosaccharide studies. None of the unsaturated disaccharides were desulfated by either enzyme. The inability of these enzymes to do so was confirmed in a related experiment in which all possible heparin disaccharides were first generated by pre-treating heparin with heparinase I and III prior to adding the sulfatases to the same reaction tube. The converse experiment was also conducted in which unfractionated heparin was preincubated with either sulfatase for an extended period of time (8 hours) followed by the addition of heparinase I and III. In this sequence, sulfatase pretreatment had no effect on the compositional profile of the heparinase-derived cleavage products.

While the above-mentioned experiments categorize both the 6-O-sulfatase and the N-sulfamidase as exolytic enzymes, they do not rule out the possibility of these two sulfatases acting on the non-reducing end of saturated, odd-numbered oligosaccharides. This possibility was addressed using a combination of two structurally-related sulfated trisaccharides H_(NS,6S)IH_(NS,6S) and H_(NS,6S)I_(2S)H_(NS,6S). Each of these trisaccharides was generated from the corresponding tetrasaccharides ΔU_(2S)H_(NS,6S) I_(±2S)H_(NS,6S) by the tandem use of the 2-O-sulfatase and the Δ4,5 glycuronidase prior to the addition of either the 6-O-sulfatase or N-sulfamidase. Desulfation was followed by MALDI-MS (FIG. 9). In this experiment, the 6-O-sulfatase was able to singularly desulfate both trisaccharides (FIG. 9C) The N-sulfamidase, however, was not able to do so (FIG. 9D), presumably because of the presence of the interfering 6-O-sulfate. The tandem use of the two enzymes (6-O-sufatase→N-sulfamidase) to doubly-desulfate either trisaccharide at the non-reducing end was also examined. The MALDI-MS results were equivocal, however, (FIG. 9E), and the possibility of such a species not sufficiently ionizing could not be ruled out.

The results presented for the exolytic desulfation of oligosaccharides at the non-reducing end are generally consistent with the substrate specificity data pertaining to desulfation of monosaccharide substrates. At the same time, the data could not rule out the possibility of the N-sulfamidase being absolutely refractory to oligosaccharides, perhaps as a result of the adjoining structure(s) at the reducing end. To resolve this, a modified approach was taken using both a different saccharide substrate (H_(NS)IH_(NS)IH_(NS)) as well as the means to detect the desulfated products. In this experiment, the trisulfated pentasaccharide was first generated by Δ4,5 glycuronidase treatment of the purified hexasaccharide ΔUH_(NS)IH_(NS)IH_(NS). Glycuronidase treatment was followed by incubation with the sulfamidase. All of the saccharides (untreated, Δ4,5 alone, Δ4,5 followed by N-sulfamidase) were fluorescently labeled at their reducing end through reductive amination. End labeling of the sugars permitted their detection by laser-induced fluorescence (LIF) following resolution of the products by capillary electrophoresis (FIG. 10). At each step in the experiment, saccharide peak assignment was inferred by observing discrete electrophoretic shifts in peak elution times as a function of exo-enzyme treatment. For example, conversion of the unsaturated hexasaccharide to a saturated pentasaccharide by glycuronidase-catalyzed uronic acid cleavage is consistent with the disappearance of peak X and the concomitant appearance of peak Y. Likewise, the N-desulfated pentasaccharide appears as a unique peak eluting at 7.6 minutes. From this analysis, it appears that the N-sulfamidase does desulfate some oligosaccharides in an exolytic fashion.

Discussion

Two additional sulfatase genes from the F. heparinum genome have been cloned. A BLASTP sequence homology search of the two flavobacterial genes against the protein database unambiguously identified both gene products as members of a large sulfatase family. Both protein sequences possess the signature PFAM sulfatase motif C/SXPXRXXXXS/TG (SEQ ID NO: 5) as well as the highly conserved sequence LTG (at the +9 through +11 positions relative to this motif). As is the case for most (if not all) other sulfatases that comprise this large enzyme family, this sulfatase domain is located in the N-terminal region of the encoded polypeptide. The three flavobacterial sulfatases (2-O-sulfatase, ORF B and ORF C gene products) share only a limited overall homology to one another. While the latter two flavobacterial enzymes reported here are structurally distinct from each other at this level, they do exhibit some sequence similarity to other select (mostly bacterial) sulfatases, however. In particular, ORF B shows a strong sequence homology (greater than 50%) to the mucin-desulfating sulfatase encoded by the enteric bacterium Prevotella strain RS2 (MdsA gene) (FIG. 3). In addition to mucin, this particular enzyme is specific for free N-acetylglucosamine-6-O-sulfate (Roberton, A. M., et al. (2000) Methods Mol Biol 125, 417-26). The two respective genes code for proteins of comparable molecular weight; they also exhibit strong homology throughout their respective sequences (and not merely biased toward the N-terminal sulfatase domain). The only substantive difference in their primary structure essentially involves a 31 amino acid hydrophilic insertion present in the flavobacterial sulfatase which is lacking in the Prevotella enzyme. In a similar vein, ORF C shows considerable homology to several putative sulfohydrolases annotated in the Pirelulla sp. genome (Glockner, F. O., et al. (2003) Proc Natl Acad Sci USA 100, 8298-303). The best sequence alignment is with a predicted heparan N-sulfamidase with which the flavobacterial ORF C gene product shares approximately 40% compositional identity and nearly 60% conserved amino acid substitutions distributed throughout most of the protein (FIG. 4).

Both putative sulfatases possess a cysteine-specific active site. It is at this conserved cysteine (and not serine) that the critical co- or post-translational oxidation to an L-C-α formylglycine (FGly) presumably occurs (Dierks, T., et al. (1998) FEBS Lett 423, 61-5 and Dierks, T., et al. (2005) Cell 121, 541-52). A prediction in favor of this covalent modification taking place when these two enzymes are recombinantly expressed in E. coli would be consistent with the functional expression of other cys-based sulfatases in the same heterologous system (Dierks, T., et al. (1998) J Biol Chem 273, 25560-4). Results for 2-O-sulfatase identified FGly formation at a corresponding cysteine and demonstrated its function in enzyme catalysis.

Beyond the predicted function, the putative function was confirmed first by examining the ability of these enzymes to act as so-called “arylsulfatases”, and second to act within the context of HSGAG degradation. To the first point, the results failed to unequivocally ascribe to either enzyme a “generic” sulfatase activity based exclusively on a commonly employed arylsulfatase assay. The fact that the ORF B and ORF C encoded enzymes are both poor arylsulfatases according to this assay; however, does not preclude them from acting on sulfated carbohydrates. In reality, many sulfatases (including those which desulfate heparin/heparan sulfate) fail to be classified as so-called “arylsulfatases” on the basis of this rather non-specific biochemical screen. At the same time, the results using more structurally directed monosaccharide substrates have unequivocally confirmed that two additional glycosaminoglycan sulfatases have been cloned. Moreover, the experiments go beyond this basic description and expound upon important structural determinants of enzyme specificity. In particular, the results presented identify the spatial orientation of the C4 hydroxyl as an additional structural determinant of substrate specificity, thus making the two flavobacterial sulfatases heparin/heparan sulfate degrading enzymes. The HSGAG specificity of this enzyme points to the likely existence of a unique flavobacterial gene (or set of genes) encoding the 6-O-desulfation of galactose/galactosamine.

The standard sulfatase assay described typically used singly sulfated, fluorogenic monosaccharides as substrates. In retrospect, these fluorogenic substrates were valuable in initially assigning enzyme function as well as defining the optimal in vitro reaction conditions by which to study their enzymology. At the same time, the potential use of these enzymes to desulfate heparin/heparan sulfate oligosaccharides in accordance with their defined substrate specificities was of interest. Central to this application is the question of the endolytic vs. exolytic potential of the two enzymes. By definition, the former mode of action would predict their ability to hydrolyze internally located sulfates within either a disaccharide or oligosaccharide chain. In the studies performed, neither enzyme was able to desulfate any of the unsaturated heparin/heparan disaccharides. Moreover, pre-treatment of unfractionated heparin with either enzyme failed to demonstrate loss of sulfates as assessed by compositional analysis following heparinase cleavage of the pre-treated polysaccharide.

On the other hand, an exolytic mode of action would require these enzymes to sequentially follow Δ4,5 glycuronidase hydrolysis of terminal uronic acids if, in fact, they are to act on the non-reducing end of these saccharides. The data presented here confirm this prediction, i.e., by demonstrating the ability of both enzymes to hydrolyze the non-reducing end of heparin-derived oligosaccharides. From the perspective of enzyme-substrate interactions, this reality places a structural constraint on the non-reducing end of the saccharide, namely a requirement of direct access to a sulfated hexosamine which is not hindered by the presence of an intervening uronate. It also imposes an apparent polarity to substrate binding within the enzyme active site. It is possible, however, that such a constraint is not absolutely imposed by the presence of the uronic acid per se (i.e., by virtue of being joined 1→4 to the sulfated hexosamine) but, more precisely, to the presence of the unsaturated bond at the C4 and C5 positions within this uronic acid. This chemical bond does impose a conformation of the sugar ring, which, in turn, restricts the relative orientation of the planar C5 carboxylate.

In addition, the F. heparinum HSGAG degradation pathway in vitro has been reconstructed through a biochemical description of the respective substrate specificities for each of the cloned enzymes. As such, the activity of these two enzymes has been placed in a sequential context related to the F. heparinum HSGAG degradation pathway as it presumably exists in vivo—i.e., a degradation pathway that begins with the heparin lyases (heparinases) and continues exolytically in the following order: 2-O-sulfatase→Δ4,5-glycuronidase→6-O-sulfatase→N-sulfamidase. Based on the results, it appears that both the 6-O-sulfatase and the N-sulfamidase also act downstream from 3-O-sulfatase activity.

Other questions related to the concerted activity of these enzymes in vivo also remain Chief among them is the question of what precise form the substrates for these end-of-the-line sulfatases actually take. Is may be reasonable to assume that the “natural” substrate for the 6-O-sulfatase and/or the N-sulfamidase are actually monosaccharides. This assumption is at least consistent with the sequentially exolytic nature of the flavobacterial HSGAG degrading pathway described. It is also in line with the HSGAG structure-activity relationships and the possibilities concerning active site architecture implied from these relationships. The ability of both these enzymes to desulfate fluorescently derivatized sugars in which the chromophore is linked 4→1 (α or β) to the adjoining hexosamine cannot be ignored. In addition, the ability of both these enzymes to act on longer oligosaccharides in a manner predicted by their substrate specificities is of practical value toward the use of these enzymes as discrete analytical tools for elucidating HSGAG fine structure.

In addition to describing the critical substrate specifity for each sulfohydrolase, important biochemical parameters related to their optimal use in vitro have been defined. These include pH and the role of divalent cations, namely calcium. In regard to pH, the slightly acidic pH optima demonstrated for both of these enzymes is consistent with observations for the pH optima of the 2-O-sulfatase (Myette, J. R., et al. (2003) J Biol Chem 278, 12157-66) and Δ4,5 glycuronidase (Myette, J. R., et al. (2002) Biochemistry 41, 7424-7434) enzymes. The flavobacterial HSGAG degrading enzymes are distinguished from their lysosomal counterparts, which, by virtue of their subcellular localization are most active at pH 4.5.

An investigation relating to enzyme catalysis has also been made with crystal structures of other members of this enzyme family, which share a relatively conserved active site and an enzyme mechanism for sulfate hydrolysis (Bond, C. S., et al. (1997) Structure 5, 277-89 and Parenti, G., et al. (1997) Curr Opin Genet Dev 7, 386-91). Local sequence alignments between each of the flavobacterial sulfatases and the three structurally defined sulfatases (human arylsulfatase A (Lukatela, G., et al. (1998) Biochemistry 37, 3654-64 and Waldow, A., et al. (1999) J Biol Chem 274, 12284-8) and B (Bond, C. S., et al. (1997) Structure 5, 277-89) and the sulfatase from P. aeriginosa (Boltes, I., et al. (2001) Structure (Camb) 9, 483-91)) suggest histidine 130 for the 6-O-sulfatase as at least one of the homologous active site residues (FIG. 11).

In addition to histidine, other residues which line this consensus active site include at least three additional basic residues, which appear to form a binding pocket of positive ions. Two of these positively charged residues interact electrostatically with the negatively charged oxyanions of the sulfate; a third appears to interact with the hydrated formylglycine via a hydrogen bond. Potential homologous positions in the primary sequences of the two flavobacterially derived enzymes are shown in FIG. 11.

In addition, a catalytic role for calcium was supported by the kinetic data and consensus structure of the enzyme active site. In a snapshot of crystallographic studies, a divalent metal ion coordinates with at least one sulfate oxygen of the substrate while also coordinating with the carboxylates of four highly conserved acid residues surrounding the modified cysteine (FGly). Presumably, this coordination would promote catalysis by properly orienting the sulfate ester/amide bond for hydrolysis.

In reporting together the cloning of both sulfatase genes and an inclusive description of their enzymology, there is a risk of deemphasizing an obvious functional distinction. N-sulfated hexosamines are unique to heparin and heparan sulfate. It follows that the catalytic mechanism of sulfamide hydrolysis must also be somewhat unique from that of the sulfate ester. This distinction holds true, even when one considers the ubiquitous involvement of a formylglycine in enzyme catalysis. It is perhaps this unique chemistry that explains why the N-sulfamidase, unlike the 6-O-sulfatase, was largely uninhibited by sulfate or phosphate ions. It may also explain other empirical distinctions observed for the two enzymes, such as the involvement of calcium, the seeming inability to hydrolyze sulfated aromatic substrates (arylsulfates), or the inhibition by the presence of secondary sulfates within the glucosamine. As form follows function, this distinction naturally plays out at the level of enzyme structure. For the lysosomal N-sulfamidase, this distinction is evident even at the primary sequence level (where there is only about 10-25% identity to O-sulfatases). This limited sequence homology generally holds true when making the same comparison between the flavobacterial enzymes.

Even when one compares the heparan sulfamidase between divergent organisms such as flavobacterium and mammals, discrete structural differences are likely given the reversed order within the degradation sequence in which the two enzymes act. In the lysosomal pathway, the N-sulfamidase is a relatively early enzyme that precedes the 6-O sulfatase, whereas our results indicate a reverse order for the flavobacterial enzymes. As such, the lysosomal heparin N-sulfamidase may naturally possess broader substrate specificity relative to the functional homologue from F. heparinum. It follows that the relative active site topologies may also differ, especially as it pertains to additional residues for the lysosomal enzyme that must accommodate secondary sulfate interactions.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

The listing of references herein is not intended to be an admission that any of the references is a prior art reference. 

What is claimed is:
 1. A method, comprising contacting a glycosaminoglycan with a glycosaminoglycan-degrading enzyme in an amount effective to cleave the glycosaminoglycan, thereby producing a cleavage product, wherein the glycosaminoglycan-degrading enzyme is an isolated sulfatase comprising the amino acid sequence of SEQ ID NO: 2 or
 4. 2. The method of claim 1, wherein the isolated sulfatase comprises the amino acid sequence of SEQ ID NO:2.
 3. The method of claim 1, wherein the isolated sulfatase comprises the amino acid sequence of SEQ ID NO:4.
 4. The method of claim 1, wherein the glycosaminoglycan is further contacted with at least one additional glycosaminoglycan-degrading enzyme.
 5. The method of claim 4, wherein the at least one additional glycosaminoglycan-degrading enzyme is used prior to, subsequent to, or concurrently with the sulfatase.
 6. The method of claim 4, wherein the at least one additional glycosaminoglycan-degrading enzyme is heparinase I, heparinse II, heparinase III, 2-O sulfatase, or Δ4,5 glycuronidase.
 7. The method of claim 4, wherein the at least one additional glycosaminoglycan-degrading enzyme is another sulfatase, glycosyl hydrolase, endoglucuronidase, or lyase.
 8. The method of claim 6, wherein the at least one additional glycosaminoglycan-degrading enzyme is Δ4,5 glycuronidase.
 9. The method of claim 8, wherein the Δ4,5 glycuronidase is contacted with the glycosaminoglycan prior to the sulfatase.
 10. The method of claim 9, wherein the glycosaminoglycan is further contacted with a 2-O sulfatase and the contact with the 2-O sulfatase is prior to the contact with the Δ4,5 glycuronidase.
 11. The method of claim 10, wherein the glycosaminoglycan is further contacted with a heparinase and the contact with the heparinase is prior to the contact with the 2-O sulfatase.
 12. The method of claim 4, wherein the sulfatase comprises the amino acid sequence of SEQ ID NO:2; wherein the glycosaminoglycan is also contacted with N-sulfamidase, and wherein the glycosaminoglycan is contacted with the sulfatase prior to the N-sulfamidase.
 13. The method of claim 11, wherein the sulfatase comprises the amino acid sequence of SEQ ID NO:2; wherein the glycosaminoglycan is also contacted with N-sulfamidase; and wherein the glycosaminoglycan is contacted with the sulfatase prior to the N-sulfamidase.
 14. The method of claim 1, wherein the glycosaminoglycan is 3-O desulfated prior to contact with the sulfatase.
 15. The method of claim 1, further comprising analyzing the cleavage product.
 16. The method of claim 15, wherein the glycosaminoglycan is also contacted with at least one additional glycosaminoglycan-degrading enzyme.
 17. The method of claim 16, wherein the at least one additional glycosaminoglycan-degrading enzyme is used prior to, subsequent to, or concurrently with the sulfatase.
 18. The method of claim 16, wherein the at least one additional glycosaminoglycan-degrading enzyme is heparinase I, heparinse II, heparinase III, 2-O sulfatase, or Δ4,5 glycuronidase.
 19. The method of claim 16, wherein the at least one additional glycosaminoglycan-degrading enzyme is another sulfatase, glycosyl hydrolase, endoglucuronidase, or lyase.
 20. The method of claim 18, wherein the at least one additional glycosaminoglycan-degrading enzyme is Δ4,5 glycuronidase.
 21. The method of claim 20, wherein the Δ4,5 glycuronidase is contacted with the glycosaminoglycan prior to the sulfatase.
 22. The method of claim 21, wherein the glycosaminoglycan is further contacted with a 2-O sulfatase and the contact with the 2-O sulfatase is prior to the contact with the Δ4,5 glycuronidase.
 23. The method of claim 22, wherein the glycosaminoglycan is further contacted with a heparinase and the contact with the heparinase is prior to the contact with the 2-O sulfatase.
 24. The method of claim 16, wherein the sulfatase comprises the amino acid sequence of SEQ ID NO:2; wherein the glycosaminoglycan is also contacted with an N-sulfamidase; and wherein the glycosaminoglycan is contacted with the sulfatase prior to the N-sulfamidase.
 25. The method of claim 23, wherein the sulfatase comprises the amino acid sequence of SEQ ID NO:2; wherein the glycosaminoglycan is also contacted with an N-sulfamidase; and wherein the glycosaminoglycan is contacted with the sulfatase prior to the N-sulfamidase.
 26. The method of claim 15, wherein the glycosaminoglycan is 3-O desulfated prior to contact with the sulfatase.
 27. The method of claim 15, wherein the presence of a particular glycosaminoglycan in a sample is determined based on the result from the analyzing step.
 28. The method of claim 15, wherein the purity of a glycosaminoglycan in a sample is determined based on the result from the analyzing step.
 29. The method of claim 15, wherein the composition of a glycosaminoglycan in a sample is determined based on the result from the analyzing step.
 30. The method of claim 15, wherein the sequence of saccharide units in a glycosaminoglycan is determined based on the result from the analyzing step.
 31. The method of claim 15, wherein the analyzing step is performed by subjecting the cleavage product to mass spectrometry, NMR spectroscopy, gel electrophoresis, capillary electrophoresis, or HPLC. 